Compositions and methods for differentiating pluripotent stem cells into primitive blood cells and uses thereof

ABSTRACT

Compositions and methods that employ various combinations of such factors as retinoic acid signaling inhibitors, antioxidants, BMP4, VEGF, prostaglandin E 2  pathway stimulants, TPO, SCF, FLT-3, EPO, TGFβ1, p38 MAPK inhibitors, beta adrenergic receptor agonists, cell cycle inhibitors, RXR agonists, Cripto, and chromatin remodelers to drive differentiation of pluripotent stem cells towards primitive blood cells. Uses of such primitive blood cells are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 USC §119(e) to U.S.Provisional Patent Application 61/566,940 filed Dec. 5, 2011, theentirety of which is incorporated herein by reference.

FIELD OF THE INVENTION

The invention is directed to compositions and methods fordifferentiating cells, such as differentiating pluripotent stem cellsinto primitive blood cells, and downstream uses thereof.

BACKGROUND

The scarcity of human embryonic tissue and the difficulty inmanipulating this tissue for research purposes has increased the use ofhuman induced pluripotent stem (iPS) cells and their in vitrodifferentiation systems. While factors that influence cell developmentand maturation have been explored, it is difficult to recapitulateembryonic development in vitro. The cell types, cellularniches/structures, and the resulting embryonic patterning are influencedby the artificial conditions in the in vitro setting, which often leadsto disorganized, semi-complete development of organ systems.

Current published in vitro protocols demonstrate very limited efficiencyin differentiating ES and iPS cells to hematopoietic lineages and, inparticular, generating significant numbers of primitive hematopoieticstem cells, hematopoietic progenitors, and hematopoietic cells. This maybe attributed partly to the inability of in vitro systems to mimic theprocesses of embryonic development towards the precursor cells of thehematopoietic system. Additionally, in vitro systems experience unwanteddifferentiation of ES and iPS cells towards non-blood lineages, such asneuroectoderm and cardiac (anterior lateral plate) mesoderm.

As a consequence, current published in vitro protocols produceinsufficient numbers of immature hematopoietic output cells to performhematopoietic stem cell (HSC) and progenitor or mature cell transplantsin patients. It also is difficult to produce enough hematopoietic stemcells, hematopoietic progenitor cells, and hematopoietic cells forresearch and laboratory use.

The ability to generate hematopoietic stem cells, hematopoieticprogenitor cells, and mature blood cells from patient derived inducedpluripotent stem (iPS) cells, would enable the generation of anunlimited supply of human leukocyte antigen (HLA)-matched transplantablecells (with or without genetic modification, such as to correct amonogenic disease); these transplantable cells would be used for thetreatment of both hematological disorders or malignancies wherehematopoietic cell transplantation is required. There is a need forcompositions and methods that produce such cells.

SUMMARY OF THE INVENTION

The present invention exploits novel pathways involved in hematopoieticstem and progenitor cell generation and expansion from human ES and iPScells. Using the components described herein, we specifically enhancethe generation of phenotypic adult hematopoietic stem cells and moreclosely mimic the developmental stages and/or niches of hematopoiesis inthe embryo, including the aorta-gonad-mesonephros (AGM) nicheenvironment, from which definitive hematopoiesis and repopulatinghematopoietic stem cells are first established in the developing embryo.

As described herein, inhibiting retinoic acid (RA) signaling increasesthe expression of factors associated with mesoderm specification duringgerm layer specification and increases hemogenic mesodermal precursorsand progenitors. In addition, inhibiting RA results in a 3-fold increasein hematopoietic cells having lymphoid and myeloid differentiationcapacity and also having an adult cell surface HSC phenotype. Thisexpansion is obtained without an increase in differentiated blood cells,indicative of increased self-renewal of these primitive multipotentblood cells.

One purpose of the invention is to better direct differentiation ofpluripotent stem cells such as ES and iPS cells towards the mesoderm andhematopoietic lineage and thereby increase the efficiency of in vitroblood cell production systems. The system described herein demonstratesthe successful recapitulation of the in vivo setting of RA roles duringmammalian and human embryonic developmental stages. These resultsindicate that RA production during development and in the adult ishighly regulated and an integral part of achieving tissue and organdevelopment. The methods described herein provide improved protocols forpluripotent stem cell differentiation to blood, including HSCs, fortransplantation into patients with hematological disorders andmalignancies.

The compositions and methods provided herein ensure that as much of thepluripotent stem cell starting material as possible reaches adevelopmental state where it carries the capacity to give rise tohematopoietic cells. To avoid losing cells to unwanted differentiation,the system minimizes unwanted disturbances that would negatively affectthe process of directed differentiation.

A version of a differentiation medium as provided herein comprises aretinoic acid signaling inhibitor, an antioxidant, and BMP4. Thedifferentiation medium may further comprise one, all, or anysubcombination of components selected from the group consisting of astimulant of prostaglandin E2 pathway, TPO, VEGF, SCF, FLT-3, EPO, andTGFβ1. The differentiation medium may alternatively or additionallyfurther comprise one, all, or any combination of components selectedfrom the group consisting of a p38 MAPK inhibitor, a beta adrenergicreceptor agonist, a cell cycle inhibitor, an RXR agonist, Cripto, and achromatin remodeler. The p38 MAPK inhibitor is preferably LY2228820. Thecell cycle inhibitor is preferably rapamycin. The RXR agonist ispreferably LG101506. The beta adrenergic receptor agonist may compriseone, all, or any combination of components selected from the groupconsisting of norepinephrine, epinephrine, salmeterol, andisoproterenol. The chromatin remodeler is preferably valproic acid. Theantioxidant may comprise one, all, or any combination of componentsselected from the group consisting of sodium selenite, butein,glutathione, and N-acetyl cysteine. The retinoic acid signalinginhibitor preferably comprises DEAB.

A version of a differentiation method as provided herein comprisesexposing a cell to a differentiation medium as described above orotherwise described herein. The cell exposed to the differentiationmedium is preferably a human cell. In certain versions, the cell isexposed to the differentiation medium in the absence of feeder cells. Insome versions, the cell preferably comprises a pluripotent stem cell,and the cell is preferably exposed to the differentiation medium for atime and under conditions sufficient to generate a hematopoietic stemcell. The hematopoietic stem cell preferably is capable of furtherdifferentiating into a myeloid cell and also is preferably capable offurther differentiating into a lymphoid cell. Exposing the cell to thedifferentiation medium preferably comprises initially exposing the cellto the differentiation medium by adding a volume of the differentiationmedium to an existing volume of non-differentiation medium to generate amixed medium, exposing the cell to the mixed medium, and subsequentlyreplacing substantially all the mixed medium after about 12-48 hoursfrom the initial exposure with a fresh volume of the differentiationmedium. After replacing substantially all the mixed medium, a freshvolume of the differentiation medium preferably is added only to anexisting volume of the differentiation medium, wherein the existingvolume comprises medium exposed to the cell for a period of at least twohours. In certain versions, the cell comprises an intact embryoid bodyformed for a period of at least 7 days prior to the exposing. The intactembryoid body preferably is exposed to the differentiation medium for aperiod of at least 5 days prior to plating the embryoid body, whichpreferably is followed by plating the embryoid body. In certainversions, the embryoid body is plated in the absence of feeder cells.After plating, a fresh volume of the differentiation medium preferablyis added only to an existing volume of the differentiation mediumwithout removing any of the existing volume of the differentiationmedium, wherein the existing volume comprises medium exposed to the cellfor a period of at least 15 minutes. Some versions of thedifferentiation method further comprise differentiating thehematopoietic stem cell into a cell selected from the group consistingof a myeloid cell and a lymphoid cell. Differentiating the hematopoieticstem cell into a myeloid cell or a lymphoid cell may be performed invitro, in vivo, or a combination thereof.

The invention further provides methods of engrafting a blood cell in ahuman subject. One version comprises administering a hematopoietic stemcell generated by a method as described above or a cell differentiatedtherefrom to the subject.

The objects and advantages of the invention will appear more fully fromthe following detailed description of the preferred embodiment of theinvention made in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B depict schematics of a differentiation pathway from embryonicstem (ES) or induced pluripotent stem (iPS) cells to hematopoietic stemcells (HSCs). FIG. 1A shows the steps affected by DEAB and the effectsresulting therefrom in accordance with a differentiation method of thepresent invention. FIG. 1B shows the steps affected by BMP4 and VEGF andthe effects resulting therefrom in accordance with a differentiationmethod of the present invention.

FIGS. 2A and B depict aspects of a differentiation method of the presentinvention. FIG. 2A depicts a schematic of the differentiation pathwayfrom embryonic stem (ES) or induced pluripotent stem (iPS) cells tohematopoietic stem cells (HSCs) with respect to a timeline of adifferentiation method of the present invention. FIG. 2B depicts a tableindicating method steps and effects thereof at select days of adifferentiation method corresponding to the method shown in FIG. 2A.

FIGS. 3A-C depicts results of a small molecule screen to identifycompounds that improve the output of phenotypic hematopoietic stem cellsand hematopoietic progenitor cells. FIG. 3A shows the fold change inphenotypic hematopoietic stem cells (CD45+, CD43+, CD34+, CD38−, CD90+,CD45RA−) for each screened compound relative to a DMSO control. FIG. 3Bshows the total hematopoietic stem cell (CD45+, CD43+, CD34+, CD38−,CD90+, CD45RA−) output resulting from each screened compound compared toa DMSO control. FIG. 3C shows the fold increase in total blood cells(CD45+, CD43+) for each screened compound relative to a DMSO control.FIG. 3D shows the fold increase in the colony/burst forming units(CFU/BFU) of myeloid hematopoietic progenitors for each screenedcompound relative to a DMSO control, depicting in detail the changes inerythroid burst-forming units (BFU-E), granulocyte colony-forming units(CFU-G), macrophage colony-forming units (CFU-M), and multi-potentialgranulocyte, erythroid, macrophage, megakaryocyte colony-forming units(CFU-GEMM).

FIGS. 4A-D depict results of iPS cells differentiated in mediacontaining DEAB. FIG. 4A shows cell counts of total viable cells, totalblood cells (CD45+, CD43+), progenitor cells (CD45+, CD43+, CD34+), andHSCs (CD45+, CD43+, CD34+, CD38−, CD90+, CD45RA−) differentiated inmedia containing DMSO (left-hand bars/non-hashed) or 10 μM DEAB(right-hand bars/hashed). FIG. 4B shows fold changes in cell counts oftotal viable cells, total blood cells (CD45+, CD43+), progenitor cells(CD45+, CD43+, CD34+), and HSCs (CD45+, CD43+, CD34+, CD38−, CD90+,CD45RA−) differentiated in media containing DMSO (left-handbars/non-hashed) or 10 μM DEAB (right-hand bars/hashed). FIG. 4C showsviable cells, total blood cells (CD45+, CD43+), progenitor cells (CD45+,CD43+, CD34+), and HSCs (CD45+, CD43+, CD34+, CD38−, CD90+, CD45RA−) asa percentage of total cells differentiated in media containing DMSO(left-hand bars/non-hashed) or 10 μM DEAB (right-hand bars/hashed). FIG.4D shows fold change in the percentage viable cells, total blood cells(CD45+, CD43+), progenitor cells (CD45+, CD43+, CD34+), and HSCs (CD45+,CD43+, CD34+, CD38−, CD90+, CD45RA−) with respect to total cellsdifferentiated in media containing DMSO (left-hand bars/non-hashed) or10 μM DEAB (right-hand bars/hashed).

FIG. 5 depicts an increase in colony counts of hematopoietic progenitorcells differentiated from iPS cells in media containing increasingconcentrations of DEAB, depicting in detail the changes in erythroidburst-forming units (BFU-E), granulocyte colony-forming units (CFU-G),macrophage colony-forming units (CFU-M), and multi-potentialgranulocyte, erythroid, macrophage, megakaryocyte colony-forming units(CFU-GEMM).

FIGS. 6A-P depict changes in gene expression at days 0, 4, 8, 12, and 15of differentiation of pluripotent stem cells to hematopoietic lineagesin the presence of DEAB in accordance with a method of the presentinvention. FIG. 6A shows expression of PAX6. FIG. 6B shows expression ofBrachyury. FIG. 6C shows expression of MIXL1. FIG. 6D shows expressionof SOX17. FIG. 6E shows expression of FOXF1. FIG. 6F shows expression ofFOXC1. FIG. 6G shows expression of NKX2.5. FIG. 6H shows expression ofAPLNR. FIG. 6I shows expression of RUNX1. FIG. 6J shows expression ofPDGFRA. FIG. 6K shows expression of FLK1. FIG. 6L shows expression ofCD31. FIG. 6M shows expression of SCL. FIG. 6N shows expression ofRARalpha. FIG. 6O shows expression of RARbeta. FIG. 6P shows expressionof CD38.

FIG. 7A-J depict flow cytometry results of cells in the lymphoid lineagedifferentiated from cord blood-derived CD45+, CD43+, CD34+ cells andiPS-derived CD45+, CD43+, CD34+. The iPS-derived CD45+, CD43+, CD34+cells used for differentiating into lymphoid cells were generated in thepresence of DEAB in accordance with a method of the present invention.FIGS. 7A and B show the population of CD33− cells (box within the heatmap graph) sorted out for further lymphoid specific analysis. As CD33+is a definitive marker for myeloid cells, these boxes representnon-myeloid (CD33−) cells presumed to be lymphoid cells differentiatedfrom cord blood-derived or iPS-derived cells, respectively. FIGS. 7C andD show levels of B-lymphoid cells (CD33−, CD56−, CD10+, CD19+)differentiated from cord blood-derived or iPS-derived cells,respectively. FIGS. 7E and F show levels of T-lymphoid cells (CD56−,CD5+, CD7+) differentiated from cord blood-derived or iPS-derived cells,respectively. FIGS. 7G and H show levels of natural killer (NK) cells(CD16+, CD56+) differentiated from cord blood-derived or iPS-derivedcells, respectively. FIGS. 7I and 7J show levels of helper T-lymphoidcells (CD56−, CD4+, CD3+) differentiated from cord blood-derived oriPS-derived cells, respectively.

FIGS. 8A and B depict summaries of flow cytometry results of cellsdifferentiated from pluripotent stem cells in the presence of DEAB withand without the p38 inhibitor LY2228820. FIG. 8A shows the percentage ofhematopoietic progenitors (CD45+, CD43+, CD34+; shown as “CD45+,CD34+”), intermediate cells between progenitor and phenotypichematopoietic stem cells (CD45+, CD43+, CD34+, CD38−; shown as “CD45+,CD38−”), and phenotypic hematopoietic stem cells (CD45+, CD43+, CD34+,CD38−, CD90+, CD45RA−; shown as “CD90+, CD45RA−”) among total cellsdifferentiated from H1 ES cells, as well as the total % increase in HSCcell number resulting from exposure to LY2228820. FIG. 8B shows thepercentage of hematopoietic progenitors (CD45+, CD43+, CD34+; shown as“CD45+, CD34+”), intermediate cells between progenitor and phenotypichematopoietic stem cells (CD45+, CD43+, CD34+, CD38−; shown as “CD45+,CD38−”), and phenotypic hematopoietic stem cells (CD45+, CD43+, CD34+,CD38−CD90+, CD45RA−; shown as “CD90+, CD45RA−”) among total cellsdifferentiated from iPS cells, as well as the total % increase in HSCcell number resulting from exposure to LY2228820.

FIGS. 9A-C depict summaries of flow cytometry results of cellsdifferentiated from human ES cells in the presence of DEAB (control)with and without various amounts of norepinephrine (10 μM or 300 μM).FIG. 9A shows fold changes in the percentage of blood cells among viablecells. FIG. 9B shows fold changes in the percentage of intermediatecells between progenitor and phenotypic hematopoietic stem cells (CD34+,CD38− cells) among total blood cells (CD45+, CD43+, CD34+, CD38−; shownas “CD34+, CD38−”). FIG. 9C shows fold changes in the percentage ofphenotypic hematopoietic stem cells (CD45+, CD43+, CD34+, CD38−, CD90+,CD45RA−; shown as “CD90+, CD45RA”) among total blood cells.

FIGS. 10A and B depict summaries of flow cytometry results of cellsdifferentiated from pluripotent stem cells in the presence of DEAB withand without LG101506. FIG. 10A shows the percentage of hematopoieticprogenitors (CD45+, CD43+, CD34+; shown as “CD45+, CD34+”), intermediatecells between progenitor and phenotypic hematopoietic stem cells (CD45+,CD43+, CD34+, CD38−; shown as “CD45+, CD38−”), and phenotypichematopoietic stem cells (CD45+, CD43+. CD34+, CD38−, CD90+, CD45RA−;shown as “CD90+, CD45RA−”) among total cells differentiated from H1 EScells, as well as the total % increase in HSC cell number resulting fromexposure to LG101506. FIG. 10B shows the percentage of hematopoieticprogenitors (CD45+, CD43+, CD34+; shown as “CD45+, CD34+”), intermediatecells between progenitor and phenotypic hematopoietic stem cells (CD45+,CD43+, CD34+, CD38−; shown as “CD45+, CD38−”), and phenotypichematopoietic stem cells (CD45+, CD43+, CD34+, CD38−CD90+, CD45RA−;shown as “CD90+, CD45RA−”) among total cells differentiated from iPScells, as well as the total % increase in HSC cell number resulting fromexposure to LG101506.

FIGS. 11A and B depict summaries of flow cytometry results of cellsdifferentiated from pluripotent stem cells in the presence of DEAB withand without sodium selenite. FIG. 11A shows the percentage ofhematopoietic progenitors (CD45+, CD43+, CD34+; shown as “CD45+,CD34+”), intermediate cells between progenitor and phenotypichematopoietic stem cells (CD45+, CD43+, CD34+, CD38−; shown as “CD45+,CD38−”), and phenotypic hematopoietic stem cells (CD45+, CD43+, CD34+,CD38−, CD90+, CD45RA−; shown as “CD90+, CD45RA−”) among total cellsdifferentiated from H1 ES cells, as well as the total % increase in HSCcell number resulting from exposure to sodium selenite. FIG. 11B showsthe percentage of hematopoietic progenitors (CD45+, CD43+, CD34+; shownas “CD45+, CD34+”), intermediate cells between progenitor and phenotypichematopoietic stem cells (CD45+, CD43+, CD34+, CD38−; shown as “CD45+,CD38−”), and phenotypic hematopoietic stem cells (CD45+, CD43+, CD34+,CD38−, CD90+, CD45RA−; shown as “CD90+, CD45RA−”) among total cellsdifferentiated from iPS cells, as well as the total % increase in HSCcell number resulting from exposure to sodium selenite.

FIGS. 12A and B depict summaries of flow cytometry results of cellsdifferentiated from pluripotent stem cells in the presence of DEAB withand without butein. FIG. 12A shows the percentage of hematopoieticprogenitors (CD45+, CD43+, CD34+; shown as “CD45+, CD34+”), intermediatecells between progenitor and phenotypic hematopoietic stem cells (CD45+,CD43+, CD34+, CD38−; shown as “CD45+, CD38−”), and phenotypichematopoietic stem cells (CD45+, CD43+, CD34+, CD38−, CD90+, CD45RA−;shown as “CD90+, CD45RA−”) among total cells differentiated from H1 EScells, as well as the total % increase in HSC cell number resulting fromexposure to butein. FIG. 12B shows the percentage of hematopoieticprogenitors (CD45+, CD43+, CD34+; shown as “CD45+, CD34+”), intermediatecells between progenitor and phenotypic hematopoietic stem cells (CD45+,CD43+, CD34+, CD38−; shown as “CD45+, CD38−”), and phenotypichematopoietic stem cells (CD45+, CD43+, CD34+, CD38−, CD90+, CD45RA−;shown as “CD90+, CD45RA−”) among total cells differentiated from iPScells, as well as the total % increase in HSC cell number resulting fromexposure to butein.

FIGS. 13A and B depict summaries of flow cytometry results of cellsdifferentiated from pluripotent stem cells in the presence of DEAB withand without valproic acid. FIG. 13A shows the percentage ofhematopoietic progenitors (CD45+, CD43+, CD34+; shown as “CD45+,CD34+”), intermediate cells between progenitor and phenotypichematopoietic stem cells (CD45+, CD43+, CD34+, CD38−; shown as “CD45+,CD38−”), and phenotypic hematopoietic stem cells (CD45+, CD43+, CD34+,CD38−, CD90+, CD45RA−; shown as “CD90+, CD45RA−”) among total cellsdifferentiated from H1 ES cells, as well as the total % increase in HSCcell number resulting from exposure to valproic acid. FIG. 13B shows thepercentage of hematopoietic progenitors (CD45+, CD43+, CD34+; shown as“CD45+, CD34+”), intermediate cells between progenitor and phenotypichematopoietic stem cells (CD45+, CD43+, CD34+, CD38−; shown as “CD45+,CD38−”), and phenotypic hematopoietic stem cells (CD45+, CD43+, CD34+,CD38−, CD90+, CD45RA−; shown as “CD90+, CD45RA−”) among total cellsdifferentiated from iPS cells, as well as the total % increase in HSCcell number resulting from exposure to valproic acid.

FIG. 14 depicts myeloid CFU cell count data for cells differentiatedfrom pluripotent stem cells (H1 ES cells and CB1 iPS cells) in thepresence of DEAB and each of Cripto (500 ng/ml), LY2228820 (shown as“LY222820”, 100 nM), Activin A, valproic acid (VPA, 1 mM), sodiumselenite (100 nM), and butein (10 μM).

DETAILED DESCRIPTION OF THE INVENTION

FIGS. 1A and B show the differentiation pathways exploited by thepresent invention in which stem cells (e.g., induced pluripotent stemcells (iPS) or embryonic stem (ES) cells) are differentiated intohematopoietic stem cells (HSCs) or hematopoietic progenitor cells. Asdepicted, stem cells sequentially differentiate into mesoderm, lateralplate mesoderm, hematopoietic mesoderm (posterior mesoderm), aorticendothelium (hemogenic endothelium), and then HSCs. A portion of theHSCs may further differentiate into progenitors. The HSCs also mayexpand to generate additional HSCs. Markers defining the above-mentionedcell types and those otherwise described herein are indicated in Table 1and are selectively shown in FIG. 1B.

TABLE 1 Cell Markers Cell Type Markers Stem cell (ES or iPS) OCT4,SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, SOX2, NANOG, DNMT3B,CRIPTO, KLF4, C-MYC, REX1 Ectoderm SOX1, Pax6 Endoderm SOX17, GATA4,SOX7 Mesoderm Brachyury, CD31 Primitive streak mesoderm MIXL1 Paraxialmesoderm FOXF1 Lateral Plate Mesoderm FOXC1 Cardiac mesoderm NKX2.5(Anterior lateral plate mesoderm) Hematopoietic mesoderm SAL1, Wnt,GATA2, SCL, FOXC1, NKX2.5 (low) (Posterior lateral plate mesoderm)Aortic endothelium APLNR, PDGFRA (low), FLK1 (Hemogenic endothelium)Hematopoietic progenitor cell (HPC) - (CD45+, CD43+, CD34+), (CD45+,CD34+), Pluripotent stem cell-derived (CD43+, CD34+), (CD45+, CD90+),(CD43+, CD90+), (CD45+, CD43+, CD90+), (CD45+, CD43+, CD34+, CD38−),(CD45+, CD34+, CD38−), (CD43+, CD34+, CD38−) Hematopoietic progenitorcell (HPC) - (CD45+, CD43+, CD34+), (CD45+, CD34+), Cord blood-derived(CD43+, CD34+), (CD45+ CD90+), (CD43+ CD90+), (CD45+, CD43+, CD90+),(CD45+, CD43+, CD34+, CD38−), (CD45+, CD34+, CD38−), (CD43+, CD34+,CD38−) Multipotent progenitor cell (MPP) - (CD45+, CD43+, CD34+, CD38−,CD90−, Cord blood derived CD45RA−), (CD43+, CD34+, CD38−, CD90−,CD45RA−), (CD45+, CD34+, CD38−, CD90−, CD45RA−), (CD45+, CD43+, CD34+,CD38−, CD90−), (CD43+, CD34+, CD38−, CD90−), (CD45+, CD34+, CD38−,CD90−), (CD45+, CD43+, CD34+, CD38−), (CD43+, CD34+, CD38−), (CD45+,CD34+, CD38−), (CD45+, CD43+, CD34+), (CD43+, CD34+), (CD45+, CD34+)Hematopoietic stem cell (HSC), adult (CD45+, CD43+, CD34+, CD90+,CD45RA−), hematopoietic stem cell, phenotypic (CD45+, CD43+, CD34+,CD90−, CD45RA−), stem cell)-Pluripotent stem cell (CD45+, CD43+, CD34+,CD90+, CD45RA−, derived CD49f+), (CD45+, CD43+, CD34+, CD90+, CD45RA−,Rhodamine (low)), (CD45+, CD43+, CD34+, CD90−), (CD45+, CD43+, CD34+,CD90+, CD45RA−, CD49f+, Rhodamine (low)), (CD45+, CD34+, CD90+,CD45RA−), (CD45+, CD34+, CD90−), (CD45+, CD34+, CD90+, CD45RA−, CD49f+),(CD45+, CD34+, CD90−), (CD45+, CD34+, CD90+, CD45RA−, Rhodamine (low)),(CD45+, CD34+, CD90−), (CD45+, CD34+, CD90+, CD45RA−. CD49f+, Rhodamine(low)), (CD43+, CD34+, CD90+, CD45RA−), (CD43+, CD34+, CD90−), (CD43+,CD34+, CD90+, CD45RA−, CD49f+), (CD43+, CD34+, CD90+, CD45RA−, Rhodamine(low)), (CD43+, CD34+, CD90+, CD45RA−, CD49f+, Rhodamine (low)), (CD45+,CD90+), (CD45+, CD90−), (CD45+, CD90+, CD49f+), (CD45+, CD90−), (CD45+,CD90+, Rhodamine (low)), (CD45+, CD90+, CD49f+, Rhodamine (low)),(CD43+, CD90+), (CD43+, CD90−), (CD43+, CD90+, CD49f+), (CD43+, CD90+,Rhodamine (low)), (CD43+, CD90+, CD49f+, Rhodamine (low)), (CD45+,CD43+, CD90+), (CD45+, CD43+, CD90−, CD45RA−), (CD45+, CD43+, CD90+,CD49f+), (CD45+, CD43+, CD90−), (CD45+, CD43+, CD90+, Rhodamine (low)),(CD45+, CD43+, CD90+, CD45RA−, CD49f+, Rhodamine (low)), (CD45+, CD43+,CD34+, CD38−, CD90+, CD45RA−), (CD45+, CD43+, CD34+, CD38−, CD90−),(CD45+, CD43+, CD34+, CD38−, CD90+, CD45RA−CD49f+), (CD45+, CD43+,CD34+, CD38−, CD90−), (CD45+, CD43+, CD34+, CD38−, CD90+, CD45RA−,Rhodamine (low)), (CD45+, CD43+, CD34+, CD38−, CD90−), (CD45+, CD43+,CD34+, CD38−, CD90+, CD45RA−, CD49f+, Rhodamine (low)), (CD45+, CD34+,CD38−, CD90+, CD45RA−), (CD45+, CD34+, CD38−, CD90−), (CD45+, CD34+,CD38−, CD90+, CD45RA−, CD49f+), (CD45+, CD34+, CD38−, CD90−), (CD45+,CD34+, CD38−, CD90+, CD45RA−, Rhodamine (low)), (CD45+, CD34+, CD38−,CD90+, CD45RA−, CD49f+, Rhodamine (low)), (CD43+, CD34+, CD38−, CD90+,CD45RA−), (CD43+, CD34+, CD38−, CD90−), (CD43+, CD34+, CD38−, CD90+,CD45RA−, CD49f+), (CD43+, CD34+, CD38−, CD90+, CD45RA−, Rhodamine(low)), (CD43+, CD34+, CD38−, CD90+, CD45RA−, CD49f+, Rhodamine (low)),(CD45+, CD38−, CD90+), (CD45+, CD38−, CD90−), (CD45+, CD38−, CD90+,CD45RA−, CD49f+), (CD45+, CD38−, CD90+, CD45RA−, Rhodamine (low)),(CD45+, CD38−, CD90+, CD45RA−, CD49f+, Rhodamine (low)), (CD43+, CD38−,CD90+), (CD43+, CD38−, CD90+, CD49f+), (CD43+, CD38−, CD90+, Rhodamine(low)), (CD43+, CD38−, CD90+, CD49f+, Rhodamine (low)), (CD43+, CD38−,CD90−), (CD45+, CD43+, CD34+ CD38−, CD90+), (CD45+, CD43+, CD34+, CD38−,CD90−), (CD45+, CD43+, CD34+, CD38−, CD90+, CD49f+), (CD45+, CD43+,CD34+, CD38−, CD90−, CD49f+), (CD45+, CD43+, CD34+, CD38−, CD90+,Rhodamine (low)), (CD45+, CD43+, CD34+, CD38−, CD90−, Rhodamine (low)),(CD45+, CD43+, CD34+, CD38−, CD90+, CD49f+, Rhodamine (low)), (CD45+,CD43+, CD34+, CD38−, CD90−, CD49f+, Rhodamine (low)) Hematopoietic stemcell (HSC, adult (CD45+, CD43+, CD34+, CD38−, CD90+, hematopoietic stemcell, phenotypic CD45RA−) or (CD45+, CD34+, CD38−, CD90+, stem cell) -Cord blood derived CD45RA−) or (CD43+, CD34+, CD38−, CD90+, CD45RA−),(CD45+, CD43+, CD34+, CD38−, CD90+, CD45RA−, CD49f+) or (CD45+, CD34+,CD38−, CD90+, CD45RA−, CD49f+) or (CD43+, CD34+, CD38−, CD90+, CD45RA−,CD49f+), (CD45+, CD43+, CD34+, CD38−, CD90+, CD45RA−, CD49f+, Rhodamine(low)) or (CD45+, CD34+, CD38−, CD90+, CD45RA−, CD49f+, Rhodamine (low))or (CD43+, CD34+, CD38−, CD90+, CD45RA−, CD49f+, Rhodamine (low)

FIG. 1A shows the effects of 4-diethylaminobenzaldehyde (DEAB) in thepresent invention. DEAB is a retinoic acid signaling inhibitor. DEABpromotes differentiation of stem cells toward mesoderm and away fromectoderm; promotes differentiation of mesoderm toward lateral platemesoderm and away from chordamesoderm, paraxial mesoderm, andintermediate mesoderm; promotes differentiation of lateral platemesoderm toward hematopoietic mesoderm and away from cardiac mesoderm(anterior mesoderm); promotes differentiation of hematopoietic mesodermtoward aortic endothelium and away from cardinal vein endothelium;promotes expansion of HSCs; and inhibits the differentiation of HSCsinto progenitor cells. DEAB also inhibits RA production, whichcontributes to DEAB's effects at some of the aforementioned steps, asshown. In addition, stem cell leukemia (SCL; also known as TAL1 (T-cellacute lymphoblastic leukemia 1)) expression also promotesdifferentiation of lateral plate mesoderm toward hematopoietic mesodermand away from cardiac mesoderm (see Ishiguro et al., Leuk Res., 2009September, 33(9):1249-1254; and Huang et al., Mol Cell Biol., 2000March, 20(6):2248-59).

Accordingly, some versions of the differentiation medium of the presentinvention include DEAB. In certain embodiments, DEAB is included in adifferentiation medium of the present invention at a concentration offrom about 0.1 to about 1,000 μM, from about 0.3 to about 300 μM, fromabout 1 to about 100 μM, from about 3 to about 30 μM, or any rangederivable therein. In certain embodiments, DEAB is included in thedifferentiation medium at a concentration of about 0.1, about 0.3, about1, about 5, about 10, about 15, about 20, about 25, about 30, about 40,about 50, about 100, about 300, or about 1,000 μM.

In certain embodiments, DEAB is included in a differentiation medium ofthe present invention in an amount sufficient to increase the totalnumber of CD45+, CD43+, CD34+, CD38−, CD90+, CD45RA− cells obtainedthrough differentiation therewith compared to the total number of CD45+,CD43+, CD34+, CD38−, CD90+, CD45RA− cells obtained throughdifferentiation with an identical differentiation medium lacking DEAB.The increase in the total number of CD45+, CD43+, CD34+, CD38−, CD90+,CD45RA− cells may be at least about 1.1-fold, about 1.25-fold, about1.5-fold, about 1.75-fold, about 2-fold, about 2.25-fold, about2.5-fold, about 2.75-fold, about 3-fold, or more.

In certain embodiments, DEAB is included in a differentiation medium ofthe present invention in an amount sufficient to increase the proportionof CD45+, CD43+, CD34+, CD38−, CD90+, CD45RA− cells obtained throughdifferentiation therewith compared to the proportion of CD45+, CD43+,CD34+, CD38−, CD90+, CD45RA− cells obtained through differentiation withan identical differentiation medium lacking DEAB. The increase in theproportion of CD45+, CD43+, CD34+, CD38−, CD90+, CD45RA− cells may be atleast about 1.1-fold, about 1.25-fold, about 1.5-fold, about 1.75-fold,about 2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about3-fold, or more.

In certain embodiments, DEAB is included in a differentiation medium ofthe present invention in an amount sufficient to increase the number ofmyeloid progenitor cells obtained through differentiation therewithcompared to the number of myeloid progenitor cells obtained throughdifferentiation with an identical differentiation medium lacking DEAB.The increase in the number of myeloid progenitor cells may be at leastabout 1.1-fold, about 1.25-fold, about 1.5-fold, about 1.75-fold, about2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3-fold,or more.

In certain embodiments, DEAB is included in a differentiation medium ofthe present invention in an amount sufficient to increase the proportionof myeloid progenitor cells obtained through differentiation therewithcompared to the proportion of myeloid progenitor cells obtained throughdifferentiation with an identical differentiation medium lacking DEAB.The increase in the number of myeloid progenitor cells may be at leastabout 1.1-fold, about 1.25-fold, about 1.5-fold, about 1.75-fold, about2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3-fold,or more. The increase in myeloid progenitor cells may include increasesin erythroid progenitors, granulocyte progenitors, macrophageprogenitors, and multi-potential granulocyte, erythroid, macrophage, andmegakaryocyte progenitors. Increases in such myeloid progenitor cellsmay be identified by testing for increases in erythroid burst-formingunits (BFU-E), granulocyte colony-forming units (CFU-G), macrophagecolony-forming units (CFU-M), and/or multi-potential granulocyte,erythroid, macrophage, megakaryocyte colony-forming units (CFU-GEMM),respectively, as shown herein.

In certain embodiments, DEAB is included in a differentiation medium ofthe present invention in an amount sufficient to generate HSCs and/orprogenitor cells which are capable of differentiating into lymphoidcells. Such lymphoid cells may include B-lymphoid cells (CD33−, CD56−,CD10+, CD19+), T-lymphoid cells (CD56−, CD5+, CD7+), natural killer (NK)cells (CD3−, CD56+), and/or helper T-lymphoid cells (CD56−, CD4+, CD3+).Determining whether HSCs and/or progenitor cells are capable ofdifferentiating into lymphoid cells can be carried out with a lymphocytedifferentiation protocol as described in Charoudeh et al., Blood, 2010,116(2):183-92, discussed in detail below.

In certain embodiments, DEAB is included in a differentiation medium ofthe present invention in an amount sufficient to increase the amount orproportion of HSCs and/or progenitor cells capable of differentiatinginto lymphoid cells compared to the amount or proportion of HSCs and/orprogenitor cells capable of differentiating into lymphoid cells obtainedwith an identical differentiation medium lacking DEAB. Such lymphoidcells may include B-lymphoid cells (CD33−, CD56−, CD10+, CD19+),T-lymphoid cells (CD56−, CD5+, CD7+), natural killer (NK) cells (CD3−,CD56+), and/or helper T-lymphoid cells (CD56−, CD4+, CD3+). Determiningan increase in the amount or proportion of HSCs and/or progenitor cellscapable of differentiating into lymphoid cells can be carried out with alymphocyte differentiation protocol as described in Charoudeh et al.,Blood, 2010, 116(2):183-92.

In certain embodiments, DEAB is included in a differentiation medium ofthe present invention in an amount sufficient to generate HSCs and/orprogenitor cells that are capable of differentiating into both lymphoidcells and myeloid cells. Such cells may include myeloid cells (CD33+)and B-lymphoid cells (CD33−, CD56−, CD10+, CD19+), T-lymphoid cells(CD56−, CD5+, CD7+), natural killer (NK) cells (CD3−, CD56+), and/orhelper T-lymphoid cells (CD56−, CD4+, CD3+). Determining whether HSCsand/or progenitor cells are capable of differentiating into lymphoidcells can be carried out with a lymphocyte differentiation protocol asdescribed in Charoudeh et al., Blood, 2010, 116(2):183-92, discussed indetail below.

In certain embodiments, DEAB is included in a differentiation medium ofthe present invention in an amount sufficient to decrease a level ofPAX6 expression in cells exposed to the medium with respect to a levelof PAX6 expression in cells exposed to an identical differentiationmedium lacking DEAB. The relative decrease in the level of PAX6expression preferably occurs on about day 4 and day 12 after exposure tothe media.

In certain embodiments, DEAB is included in a differentiation medium ofthe present invention in an amount sufficient to increase a level ofBrachyury expression in cells exposed to the medium with respect to alevel of Brachyury expression in cells exposed to an identicaldifferentiation medium lacking DEAB. The relative increase in the levelof Brachyury expression preferably occurs on about day 4 after exposureto the media.

In certain embodiments, DEAB is included in a differentiation medium ofthe present invention in an amount sufficient to increase a level ofMIXL.1 expression in cells exposed to the medium with respect to a levelof MIXL.1 expression in cells exposed to an identical differentiationmedium lacking DEAB. The relative increase in the level of MIXL.1expression preferably occurs on about day 4 after exposure to the media.

In certain embodiments, DEAB is included in a differentiation medium ofthe present invention in an amount sufficient to decrease a level ofSOX17 expression in cells exposed to the medium with respect to a levelof SOX17 expression in cells exposed to an identical differentiationmedium lacking DEAB. The relative decrease in the level of SOX17expression preferably occurs on about day 4 after exposure to the media.

In certain embodiments, DEAB is included in a differentiation medium ofthe present invention in an amount sufficient to increase FOXF1expression in cells exposed to the medium with respect to a level ofFOXF1 expression in cells exposed to an identical differentiation mediumlacking DEAB. The relative decrease in the level of FOXF1 expressionpreferably occurs on about day 4 after exposure to the media.

In certain embodiments, DEAB is included in a differentiation medium ofthe present invention in an amount sufficient to decrease a level ofFOXC1 expression in cells exposed to the medium with respect to a levelof FOXC1 expression in cells exposed to an identical differentiationmedium lacking DEAB. The relative decrease in the level of FOXC1expression preferably occurs on about day 8 after exposure to the media.

In certain embodiments, DEAB is included in a differentiation medium ofthe present invention in an amount sufficient to decrease a level ofNKX2.5 expression in cells exposed to the medium with respect to a levelof NKX2.5 expression in cells exposed to an identical differentiationmedium lacking DEAB. The relative decrease in the level of NKX2.5expression preferably occurs on about day 8 after exposure to the media.

In certain embodiments, DEAB is included in a differentiation medium ofthe present invention in an amount sufficient to decrease a level ofAPLNR expression in cells exposed to the medium with respect to a levelof APLNR expression in cells exposed to an identical differentiationmedium lacking DEAB. The relative decrease in the level of APLNRexpression preferably occurs on about day 12 after exposure to themedia.

In certain embodiments, DEAB is included in a differentiation medium ofthe present invention in an amount sufficient to decrease a level ofPDGFRA expression in cells exposed to the medium with respect to a levelof PDGFRA expression in cells exposed to an identical differentiationmedium lacking DEAB. The relative decrease in the level of PDGFRAexpression preferably occurs on about day 12 after exposure to themedia.

In certain embodiments, DEAB is included in a differentiation medium ofthe present invention in an amount sufficient to increase a level ofFLK1 expression in cells exposed to the medium with respect to a levelof FLK1 expression in cells exposed to an identical differentiationmedium lacking DEAB. The relative increase in the level of FLK1expression preferably occurs on about day 15 after exposure to themedia.

In certain embodiments, DEAB is included in a differentiation medium ofthe present invention in an amount sufficient to increase a level ofRUNX1 expression in cells exposed to the medium with respect to a levelof RUNX1 expression in cells exposed to an identical differentiationmedium lacking DEAB. The relative increase in the level of RUNX1expression preferably occurs on about day 15 after exposure to themedia.

In certain embodiments, DEAB is included in a differentiation medium ofthe present invention in an amount sufficient to increase a level of SCLexpression in cells exposed to the medium with respect to a level of SCLexpression in cells exposed to an identical differentiation mediumlacking DEAB. The relative increase in the level of SCL expressionpreferably occurs on about day 15 after exposure to the media.

In certain embodiments, DEAB is included in a differentiation medium ofthe present invention in an amount sufficient to decrease a level ofRARbeta expression in cells exposed to the medium with respect to alevel of RARbeta expression in cells exposed to an identicaldifferentiation medium lacking DEAB. The relative decrease in the levelof RARbeta expression preferably occurs on about days 8 and/or 12 afterexposure to the media.

In certain embodiments, DEAB is included in a differentiation medium ofthe present invention in an amount sufficient to decrease a level of agene upregulated by retinoic acid in cells exposed to the medium withrespect to a level of the gene in cells exposed to an identicaldifferentiation medium lacking DEAB. An exemplary gene upregulated byretinoic acid exposure includes CD38. The relative decrease in the levelof the gene upregulated by retinoic acid preferably occurs on about days8, 12, and/or 15 after exposure to the media.

In certain embodiments, DEAB is included in a differentiation medium ofthe present invention in an amount sufficient to increase the numberand/or proportion of pluripotent stem cells differentiating to mesodermwhen exposed to the medium with respect to the number and/or proportionof pluripotent stem cells differentiating to mesoderm when exposed to anidentical differentiation medium lacking DEAB. Such a relative increasein the number and/or proportion of pluripotent stem cellsdifferentiating to mesoderm can be identified by a DEAB-dependentincrease in expression level of MIXL1 at about day 4 after exposure tothe media, a DEAB-dependent increase in expression level of Brachyury atabout day 4 and/or day 8 after exposure to the media, a DEAB-dependentdecrease in expression level of PAX6 at about day 4 after exposure tothe media, and/or a DEAB-dependent decrease in expression level of SOX17at about day 4 after exposure to the media.

In certain embodiments, DEAB is included in a differentiation medium ofthe present invention in an amount sufficient to increase the numberand/or proportion of mesoderm cells differentiating to lateral platemesoderm when exposed to the medium with respect to the number and/orproportion of mesoderm cells differentiating to lateral plate mesodermwhen exposed to an identical differentiation medium lacking DEAB. Such arelative increase in the number and/or proportion of mesoderm cellsdifferentiating to lateral plate mesoderm can be identified by aDEAB-dependent increase in expression level of FOXF1 at about day 4after exposure to the media and/or a DEAB-dependent decrease inexpression level of FOXC1 at about day 8 after exposure to the media.

In certain embodiments, DEAB is included in a differentiation medium ofthe present invention in an amount sufficient to increase the numberand/or proportion of lateral plate mesoderm cells differentiating toposterior mesoderm when exposed to the medium with respect to the numberand/or proportion of lateral plate mesoderm cells differentiating toposterior mesoderm when exposed to an identical differentiation mediumlacking DEAB. Such a relative increase in the number and/or proportionof lateral plate mesoderm cells differentiating to anterior mesoderm canbe identified by a DEAB-dependent increase in expression level of NKX2.5at about day 8 after exposure to the media.

In certain embodiments, DEAB is included in a differentiation medium ofthe present invention in an amount sufficient to decrease the numberand/or proportion of lateral plate mesoderm cells differentiating toanterior mesoderm when exposed to the medium with respect to the numberand/or proportion of lateral plate mesoderm cells differentiating toanterior mesoderm when exposed to an identical differentiation mediumlacking DEAB. Such a relative decrease in the number and/or proportionof lateral plate mesoderm cells differentiating to anterior mesoderm canbe identified by a DEAB-dependent decrease in expression level of APLNRat about day 8 after exposure to the media.

In certain embodiments, DEAB is included in a differentiation medium ofthe present invention in an amount sufficient to increase the numberand/or proportion of lateral plate mesoderm cells differentiating toposterior mesoderm when exposed to the medium with respect to the numberand/or proportion of lateral plate mesoderm cells differentiating toposterior mesoderm when exposed to an identical differentiation mediumlacking DEAB. Such a relative increase in the number and/or proportionof lateral plate mesoderm cells differentiating to posterior mesodermcan be identified by a DEAB-dependent decrease in expression level ofNKX2.5 at about day 8 after exposure to the media.

In certain embodiments, DEAB is included in a differentiation medium ofthe present invention in an amount sufficient to generate definitiveHSCs when pluripotent stem cells are contacted with the medium. As usedherein, “definitive” refers to the ability of an HSC to differentiateinto all types blood cell, i.e., the HSC is not incapable of making anyparticular type of blood cell. Exemplary types of blood cells includelymphoid cells and myeloid cells. Exemplary lymphoid cells includeB-lymphoid cells (CD33−, CD56−, CD10+, CD19+), T-lymphoid cells (CD56−,CD5+, CD7+), natural killer (NK) cells (CD3−, CD56+), and/or helperT-lymphoid cells (CD56−, CD4+, CD3+). Exemplary myeloid cells includeerythrocytes, granulocytes, monocytes, and megakaryocytes. DefinitiveHSCs can be identified by differentiating the HSCs into the above celltypes, preferably, in combination with expression of RUNX1, a definitivehematopoietic cell marker, at about day 15 after exposure to the media.

In certain embodiments, DEAB is included in a differentiation medium ofthe present invention in an amount sufficient to decrease a level ofCD31 and/or SCL expression in cells exposed to the medium with respectto a level of CD31 and/or SCL expression, respectively, in cells exposedto an identical differentiation medium lacking DEAB. CD31 and/or SCL areindicators of precursors, progenitors, and/or cells of both primitiveand definitive hematopoiesis and hemangioblast activity. Primitivehematopoiesis refers to an early wave (“primitive wave”) of bloodformation during development that is known to take place in the yolksacand is generally considered to occur outside of the AGM region duringdevelopment. This primitive wave generates hemangioblasts from precursorcells. Hemangioblasts have limited differentiation ability, arerestricted largely to the myeloid lineage, have lower proliferationcapacity, and produce erythroid cells that express embryonic and fetalhemoglobins and show limited enucleation. The relative decrease in thelevel of CD31 and/or SCL expression preferably occurs on about day 8after exposure to the media, when the primitive wave of hematopoiesisoccurs. High levels of CD31 and/SCL expression without DEAB at day 8 isindicative of hemangioblast activity and primitive (not definitive)hematopoiesis at this time point.

In certain embodiments of a differentiation medium of the presentinvention, other retinoic acid (RA) signaling inhibitors may replace orbe included with DEAB at the above-described amounts or concentrations.These other RA signaling inhibitors may act upstream or downstream ofthe RARa, RARb, or RARX receptor to inhibit signaling therefrom. Theother RA signaling inhibitors may also inhibit RA production. Exemplaryretinoic acid signaling inhibitors include disulfiram (tetraethylthiuramdisulfide or bis(diethylthiocarbamyoyl) disulfide), retinoic acidreceptor-β inhibitor LE135, the pan RA antagonist AGN 193109, andagonists of CYP26 or other molecules that aid in RA degradation.

FIG. 1B shows the role of bone morphogenetic protein-4 (BMP4) andvascular endothelial growth factor (VEGF) in the present invention. BMP4promotes differentiation of mesoderm toward lateral plate mesoderm andaway from chordamesoderm, paraxial mesoderm, and intermediate mesoderm.VEGF promotes differentiation of hematopoietic mesoderm toward aorticendothelium and away from cardinal vein endothelium. It is thought thatVEGF, at least in part, carries out its effects by Notch signaling.

Accordingly, some versions of the differentiation medium of the presentinvention include BMP4. BMP4 is a member of the group of bonemorphogenic proteins and is a ventral mesoderm inducer. BMPs areexpressed in adult human bone marrow (BM) and are important for boneremodeling and growth. In differentiation media described herein, BMP4biases differentiation towards lateral plate mesoderm and away fromchordamesoderm, paraxial mesoderm, or intermediate mesoderm, therebyenhancing generation of HSCs. See FIG. 1B. BMP4 promotes mesodermformation from pluripotent stem cells by inducing expression ofprimitive streak and early hematopoietic mesoderm genes, such asBrachyury and MIXL1, and by activating SMAD. BMP4 also enhancesprogenitor self-renewal and development of primitive hematopoieticcells. In certain embodiments, BMP4 is included in a differentiationmedium of the present invention at a concentration of from about 0.1 toabout 1,000 ng/mL, from about 0.3 to about 300 ng/mL, from about 1 toabout 100 ng/mL, from about 3 to about 30 ng/mL, or any range derivabletherein. In certain embodiments, BMP-4 is included in thedifferentiation medium at a concentration of about 0.1, about 0.3, about1, about 5, about 10, about 15, about 20, about 25, about 30, about 40,about 50, about 100, about 300, or about 1,000 ng/mL.

Some versions of the differentiation medium of the present inventioninclude vascular endothelial growth factor (VEGF). VEGF is an importantsignaling protein that is involved in formation of the embryoniccirculatory system and angiogenesis. VEGF can affect a variety of celltypes including vascular endothelium and other cell types (e.g.,neurons, cancer cells, kidney epithelial cells). In vitro, VEGF canstimulate endothelial cell mitogenesis and cell migration. VEGF functionalso has been shown to be important in a variety of disease statesincluding cancer, diabetes, autoimmune diseases, and ocular vasculardiseases. In the present system, VEGF increases definitive hematopoieticprogenitor and HSC output. In certain embodiments, VEGF is included in adifferentiation medium of the present invention at a concentration offrom about 0.01 to about 100 ng/mL, from about 0.03 to about 30 ng/mL,from about 0.1 to about 10 ng/mL, from about 0.3 to about 3 ng/mL, orany range derivable therein. In certain embodiments, VEGF is included inthe differentiation medium at a concentration of about 0.01, about 0.03,about 0.1, about 0.2, about 0.4, about 0.6, about 0.8, about 1, about1.2, about 1.4, about 1.6, about 1.8, about 3, about 10, about 15, about20, about 30, about 40, about 50, about 60, about 70, about 80, about90, or about 100 ng/mL.

Some versions of the differentiation medium of the present inventioninclude stem cell factor (also known as SCF, kit-ligand, KL, or steelfactor). SCF is a cytokine that plays a role in hematopoiesis. SCFenhances survival, proliferation, and differentiation of hematopoieticstem cells and lineage-committed hematopoietic progenitor cells. Incertain embodiments, SCF is included in a differentiation medium of thepresent invention at a concentration of from about 0.2 to about 2,000ng/mL, from about 0.6 to about 600 ng/mL, from about 2 to about 200ng/mL, from about 6 to about 60 ng/mL, or any range derivable therein.In certain embodiments, SCF is included in a differentiation medium ofthe present invention at a concentration of about 0.2, about 0.6, about1, about 5, about 10, about 15, about 20, about 25, about 30, about 35,about 40, about 50, about 60 about 200, about 600, or about 2,000 ng/mL.

Some versions of the differentiation medium of the present inventioninclude thrombopoietin (TPO). TPO is a lineage specific growth factorthat plays a role in the proliferation and maturation of megakaryocytes.TPO regulates cell cycle inhibitors Cdkn1c (p57) and Cdkn2d (p19) andhelps to maintain HSC quiescence. When included with SCF and FLT3, TPOmay expand and differentiate HSCs and progenitors. In certainembodiments, TPO is included in a differentiation medium of the presentinvention at a concentration of from about 0.2 to about 2,000 ng/mL,from about 0.6 to about 600 ng/mL, from about 2 to about 200 ng/mL, fromabout 6 to about 60 ng/mL, or any range derivable therein. In certainembodiments, TPO is included in a differentiation medium of the presentinvention at a concentration of about 0.2, about 0.6, about 1, about 5,about 10, about 15, about 20, about 25, about 30, about 35, about 40,about 50, about 60 about 200, about 600, or about 2,000 ng/mL.

Some versions of the differentiation medium of the present inventioninclude fms-related tyrosine kinase 3 ligand (FLT3). FLT3 aids inexpansion and differentiation of HSCs. FLT3 synergizes with otherfactors and interleukins and regulates proliferation of earlyhematopoietic cells. In certain embodiments, FLT3 is included in adifferentiation medium of the present invention at a concentration offrom about 0.2 to about 2,000 ng/mL, from about 0.6 to about 600 ng/mL,from about 2 to about 200 ng/mL, from about 6 to about 60 ng/mL, or anyrange derivable therein. In certain embodiments, FLT3 is included in adifferentiation medium of the present invention at a concentration ofabout 0.2, about 0.6, about 1, about 5, about 10, about 15, about 20,about 25, about 30, about 35, about 40, about 50, about 60, about 200,about 600, or about 2,000 ng/mL.

Some versions of the differentiation medium of the present inventioninclude erythropoietin (EPO). EPO promotes erythrocyte formation andprevents apoptosis. EPO also promotes HSC maintenance. In certainembodiments, EPO is included in a differentiation medium of the presentinvention at a concentration of from about 0.2 to about 2,000 ng/mL,from about 0.6 to about 600 ng/mL, from about 2 to about 200 ng/mL, fromabout 6 to about 60 ng/mL, or any range derivable therein. In certainembodiments, EPO is included in a differentiation medium of the presentinvention at a concentration of about 0.2, about 0.6, about 1, about 5,about 10, about 15, about 20, about 25, about 30, about 35, about 40,about 50, about 60 about 200, about 600, or about 2,000 ng/mL. In someversions, high doses of thrombopoietin may be included in place of EPO.

Some versions of the differentiation medium of the present inventioninclude transforming growth factor beta (TGFβ1). TGFβ1 increases HSCyield by increasing mesoderm specification, promotes HSC quiescence,upregulates p57 cyclin-dependent kinase inhibitor, modulates SMADsignaling induced by BMP4, and enhances total blood output. In certainembodiments, TGFβ1 is included in a differentiation medium of thepresent invention at a concentration of from about 0.05 to about 500ng/mL, from about 0.15 to about 150 ng/mL, from about 0.5 to about 50ng/mL, from about 1.5 to about 15 ng/mL, or any range derivable therein.In certain embodiments, TGFβ1 is included in a differentiation medium ofthe present invention at a concentration of about 0.05, about 0.15,about 0.5, about 1, about 2.5, about 5, about 7.5, about 10, about 15,about 20, about 30, about 40, about 50, about 100, about 150, or about500 ng/mL.

Some versions of the differentiation medium of the present inventioninclude rapamycin. Rapamycin is an inhibitor of the mTOR pathway.Rapamycin is a cell cycle inhibitor that induces HSC quiescence andlimits proliferation and differentiation of the cells. As shown in FIGS.3A-3D, rapamycin also increases yields of blood cells (CD45+, CD43+),HSCs (CD45+, CD43+, CD34+, CD38−, CD90+, CD45RA−), and myeloidprogenitors differentiated from pluripotent stem cells. In certainembodiments, rapamycin is included in a differentiation medium of thepresent invention at a concentration of from about 1 to about 10,000 nM,from about 3 to about 3,000 nM, from about 10 to about 1,000 nM, fromabout 30 to about 300 nM, or any range derivable therein. In certainembodiments, rapamycin is included in the differentiation medium at aconcentration of about 1, about 3, about 10, about 50, about 60, about70, about 80, about 90, about 100, about 110, about 120, about 130,about 140, about 150, about 300, about 1,000, about 3,000 or about10,000 nM. In some versions, other cell cycle inhibitors may be includedin place of rapamycin or in addition to rapamycin at the above-describedconcentrations. Cell cycle inhibitors are well known in the art. See,e.g., Murphey et al. Chem Biol Drug Des., 2006 68(4):213-9.

Some versions of the differentiation medium of the present inventioninclude norepinephrine. Norepinephrine is a beta adrenergic receptoragonist. Norepinephrine can serve as a maturing signal for HSCs torespond to the sympathetic nervous system. In the media describedherein, norepinephrine increases HSC output. In certain embodiments,norepinephrine is included in a differentiation medium of the presentinvention at a concentration of from about 3 to about 30,000 μM, fromabout 10 to about 10,000 μM, from about 30 to about 3,000 μM, from about10 to about 1,000 μM, or any range derivable therein. In certainembodiments, norepinephrine is included in the differentiation medium ata concentration of about 1, about 3, about 10, about 50, about 100,about 200, about 250, about 300, about 350, about 400, about 500, about1,000, about 10,000, or about 30,000 μM. In certain embodiments,norepinephrine is included in the differentiation medium at aconcentration sufficient to increase the total number of cells derivedfrom pluripotent stem cells exposed to the medium compared to the numberof cells derived from pluripotent stem cells exposed to an identicaldifferentiation medium lacking norepinephrine. In certain embodiments,norepinephrine is included in a differentiation medium of the presentinvention in an amount sufficient to increase the number and/orproportion of pluripotent stem cells differentiating to hematopoieticstem cells when exposed to the medium with respect to the number and/orproportion of pluripotent stem cells differentiating to hematopoieticstem cells when exposed to an identical differentiation medium lackingnorepinephrine. In some versions, other beta adrenergic receptoragonists, such as epinephrine, salmeterol, and/or isoproterenol, may beincluded in place of norepinephrine or in addition to norepinephrine atthe above-described concentrations.

Some versions of the differentiation medium of the present inventioninclude LG101506. LG101506 is a selective RXR agonist that alters thecellular response to retinoic acid signaling by activating RXRPPARgamma, RXR PPARalpha, and RXR PPARdelta heterodimers. LG101506increases the output of CD90+ HSCs generated from pluripotent stemcells, increases the number and proportion of CD38− cells, and enhancesCFU cellularity and output. In certain embodiments, LG101506 is includedin a differentiation medium of the present invention at a concentrationof from about 0.01 to about 100 μM, from about 0.03 to about 30 μM, fromabout 0.1 to about 10 μM, from about 0.3 to about 3 μM, or any rangederivable therein. In certain embodiments, LG101506 is included in thedifferentiation medium at a concentration of about 0.01, about 0.03,about 0.1, about 0.2, about 0.4, about 0.6, about 0.8, about 1, about1.2, about 1.4, about 1.6, about 1.8, about 3, about 10, about 15, about20, about 30, about 40, about 50, about 60, about 70, about 80, about90, or about 100 μM. In certain embodiments, LG101506 is included in thedifferentiation medium at a concentration sufficient to increase thetotal number of cells derived from pluripotent stem cells exposed to themedium compared to the number of cells derived from pluripotent stemcells exposed to an identical differentiation medium lacking LG101506.

Some versions of the differentiation medium of the present inventioninclude valproic acid. Valproic acid, a chromatin remodeler, is aninhibitor of histone deacetylases (HDAC) that affects chromatinremodeling by maintaining open chromatin. Valproic acid blocks HSCdifferentiation and promotes primitive HSC maintenance and repopulatingability. In the present system, valproic acid increases the proportionof CD34+, CD38− cells that are CD90+. In certain embodiments, valproicacid is included in a differentiation medium of the present invention ata concentration of from about 0.01 to about 100 mM, from about 0.03 toabout 30 mM, from about 0.1 to about 10 mM, from about 0.3 to about 3mM, or any range derivable therein. In certain embodiments, valproicacid is included in the differentiation medium at a concentration ofabout 0.01, about 0.03, about 0.1, about 0.2, about 0.4, about 0.6,about 0.8, about 1, about 1.2, about 1.4, about 1.6, about 1.8, about 3,about 10, about 15, about 20, about 30, about 40, about 50, about 60,about 70, about 80, about 90, or about 100 mM. In certain embodiments,valproic acid is included in the differentiation medium at aconcentration sufficient to increase the total number of cells derivedfrom pluripotent stem cells exposed to the medium compared to the numberof cells derived from pluripotent stem cells exposed to an identicaldifferentiation medium lacking valproic acid. In certain embodiments,valproic acid is included in a differentiation medium of the presentinvention in an amount sufficient to increase the number and/orproportion of pluripotent stem cells differentiating to hematopoieticstem cells when exposed to the medium with respect to the number and/orproportion of pluripotent stem cells differentiating to hematopoieticstem cells when exposed to an identical differentiation medium lackingvalproic acid. In some versions, other chromatin remodelers, such aschlamodicin, sodium buterate, mocetinostat, NVP-LAQ824, Mi-2beta,inositol, vorinostat, may be included with or in place of valproic acidat the above-described concentrations.

Some versions of the differentiation medium of the present inventioninclude Cripto. Cripto is a protein expressed in hypoxic endosteal nichecells. Cripto promotes primitive HSC development and regulates HSCquiescence. Cripto induces HSCs to respond as though they are in ahypoxic environment. In the present system, Cripto increases the size ofCFU colonies. In certain embodiments, Cripto is included in adifferentiation medium of the present invention at a concentration offrom about 5 to about 50,000 ng/mL, from about 15 to about 15,000 ng/mL,from about 50 to about 5.000 ng/mL, from about 150 to about 1,500 ng/mL,or any range derivable therein. In certain embodiments, Cripto isincluded in a differentiation medium of the present invention at aconcentration of about 5, about 15, about 50, about 100, about 200,about 300, about 400, about 500, about 600, about 700, about 800, about900, about 1,000, about 1,500, about 5,000, about 15,000 ng/mL, or about50,000 ng/mL.

Some versions of the differentiation medium of the present inventioninclude the p38 mitogen-activated protein kinase (p38 MAPK) inhibitorLY2228820. LY2228820 indirectly inhibits NF-κB signaling, reducescellular inflammation responses, and reduces cellular damage and stress.In the present system, LY2228820 results in an increase of the mostprimitive fraction of blood cells (CD90+) and increases CFU size. Incertain embodiments, LY2228820 is included in a differentiation mediumof the present invention at a concentration of from about 1 to about10,000 nM, from about 3 to about 3,000 nM, from about 10 to about 1,000nM, from about 30 to about 300 nM, or any range derivable therein. Incertain embodiments, LY2228820 is included in the differentiation mediumat a concentration of about 1, about 3, about 10, about 50, about 60,about 70, about 80, about 90, about 100, about 110, about 120, about130, about 140, about 150, about 300, about 1,000, about 3,000 or about10,000 nM. In certain embodiments, LY2228820 is included in thedifferentiation medium at a concentration sufficient to increase thetotal number of cells derived from pluripotent stem cells exposed to themedium compared to the number of cells derived from pluripotent stemcells exposed to an identical differentiation medium lacking LY2228820.In certain embodiments, LY2228820 is included in a differentiationmedium of the present invention in an amount sufficient to increase thenumber and/or proportion of pluripotent stem cells differentiating tohematopoietic stem cells when exposed to the medium with respect to thenumber and/or proportion of pluripotent stem cells differentiating tohematopoietic stem cells when exposed to an identical differentiationmedium lacking LY2228820. In some versions, other p38 MAPK inhibitors,such as SB 203580, BIRB 796 (Doramapimod), SB 202190, VX-702, VX-745, orPH-797804, may be included with or in place of LY2228820 at theabove-described concentrations.

Some versions of the differentiation medium of the present inventioninclude one or more antioxidants that protect against stress fromreactive oxygen species. The inventors have found that simple culturedifferentiation of pluripotent cells results in many double strand (DS)DNA breaks, as marked by phosphorylated histone H2A variant gamma(gH2AX) staining. DS DNA breaks reduce transplant efficiency ofrepopulating HSCs. Inhibiting DNA damage of the DNA damage responseresulting from reactive oxygen species or other sources of damage(radiation, chemical, etc.) in the present pluripotent differentiationsystem will increase hematopoietic cell number, repopulating ability,and cell differentiation potential. Preferred antioxidants that may beincluded in the medium of the present invention comprise ascorbic acid,NAC (N-Acetyl Cysteine), glutathione, sodium selenite, and butein,either alone or in combination.

In certain embodiments, sodium selenite is included in a differentiationmedium of the present invention at a concentration of from about 1 toabout 10,000 nM, from about 3 to about 3,000 nM, from about 10 to about1,000 nM, from about 30 to about 300 nM, or any range derivable therein.In certain embodiments, sodium selenite is included in thedifferentiation medium at a concentration of about 1, about 3, about 10,about 50, about 60, about 70, about 80, about 90, about 100, about 110,about 120, about 130, about 140, about 150, about 300, about 1,000,about 3,000 or about 10,000 nM. In certain embodiments, sodium seleniteis included in the differentiation medium at a concentration sufficientto increase the total number of cells derived from pluripotent stemcells exposed to the medium compared to the number of cells derived frompluripotent stem cells exposed to an identical differentiation mediumlacking sodium selenite.

In certain embodiments, butein is included in a differentiation mediumof the present invention at a concentration of from about 0.1 to about1,000 μM, from about 0.3 to about 300 μM, from about 1 to about 100 μM,from about 3 to about 30 μM, or any range derivable therein. In certainembodiments, butein is included in the differentiation medium at aconcentration of about 0.1, about 0.3, about 1, about 5, about 10, about15, about 20, about 25, about 30, about 40, about 50, about 100, about300, or about 1,000 μM. In certain embodiments, butein is included inthe differentiation medium at a concentration sufficient to increase thetotal number of cells derived from pluripotent stem cells exposed to themedium compared to the number of cells derived from pluripotent stemcells exposed to an identical differentiation medium lacking butein.

In certain embodiments, NAC is included in a differentiation medium ofthe present invention at a concentration of from about 0.2 to about2,000 mM, from about 0.6 to about 600 mM, from about 2 to about 200 mM,from about 6 to about 60 mM, or any range derivable therein. In certainembodiments, NAC is included in a differentiation medium of the presentinvention at a concentration of about 0.2, about 0.6, about 1, about 5,about 10, about 15, about 20, about 25, about 30, about 35, about 40,about 50, about 60 about 200, about 600, or about 2,000 mM.

In certain embodiments, ascorbic acid is included in a differentiationmedium of the present invention at a concentration of from about 0.5 toabout 5,000 ng/mL, from about 1.5 to about 1,500 ng/mL, from about 5 toabout 500 ng/mL, from about 15 to about 150 ng/mL, or any rangederivable therein. In certain embodiments, Cripto is included in adifferentiation medium of the present invention at a concentration ofabout 0.5, about 1.5, about 5, about 10, about 20, about 30, about 40,about 50, about 60, about 70, about 80, about 90, about 100, about 150,about 500, about 1,500 ng/mL, or about 5,000 ng/mL. In certain versions,other reactive oxygen species may be included in place of ascorbic acidat the above-described concentrations. These includeN-tert-butyl-α-phenylnitrone (PBN),4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL),diphenyliodonium (DPI), propyl gallate (PG), octyl gallate (OG),nordihydroguaiaretic acid (NDGA), tert-butylhydroquinone (TBHQ),ethoxyquin, 2,6-di-tert-butyl-4-hydroxymethyl-phenol (Ionox 100), orbutylated hydroxyanisole (BHA).

In certain embodiments, any one or combination of Cripto, LY2228820,valproic acid, sodium selenite, butein, or alternatives thereof areincluded in a differentiation medium of the present invention in anamount sufficient to increase the number and/or proportion of quiescenthematopoietic stem cells when exposed to the medium with respect to thenumber and/or proportion of quiescent hematopoietic stem cells whenexposed to an identical differentiation medium lacking the any one orcombination of Cripto, LY2228820, valproic acid, sodium selenite,butein, or alternatives thereof.

Some versions of the differentiation medium of the present inventioninclude one or more agents that stimulate the prostaglandin E₂ pathwayto promote HSC self-renewal and to increase RUNX1 expression. Apreferred agent is prostaglandin E₂. In certain embodiments,prostaglandin E₂ is included in a differentiation medium of the presentinvention at a concentration of from about 0.02 to about 200 μM, fromabout 0.06 to about 60 μM, from about 0.2 to about 20 μM, from about 0.6to about 6 μM, or any range derivable therein. In certain embodiments,prostaglandin E₂ is included in a differentiation medium of the presentinvention at a concentration of about 0.02, about 0.06, about 0.1, about0.5, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about4, about 5, about 6 about 20, about 60, or about 200 μM. In certainversions, other agents that stimulate the prostaglandin E₂ pathway maybe included in place of prostaglandin E₂ at the above-describedconcentrations. These include PGE2, PGI2, linoleic acid, 13(s)-HODE,LY171883, mead acid, eicosatrienoic acid, epoxyeicosatrienoic acid,ONO-259, Cay1039, 19(R)-hydroxy PGE2, 16,16-dimethyl PGE2p-(p-acetamidobenzamido) phenyl ester, 11-deoxy-16,16-dimethylPGE2,9-deoxy-9-methylene-16,16-dimethyl PGE2, 9-deoxy-9-methylene PGE2,butaprost, sulprostone, PGE2 serinol amide, PGE2 methyl ester, 16-phenyltetranor PGE2,15(S)-15-methyl PGE2, 15(R)-15-methyl PGE2, BIO,8-bromo-cAMP, forskolin, bapta-AM, fendiline, nicardipine, nifedipine,pimozide, strophanthidin, lanatoside, L-Arg, sodium nitroprusside,sodium vanadate, bradykinin, mebeverine, flurandrenolide, atenolol,pindolol, gaboxadol, kynurenic acid, hydralazine, thiabendazole,bicuculline, vesamicol, peruvoside, imipramine, chlorpropamide,1,5-pentamethylenetetrazole, 4-aminopyridine, diazoxide, benfotiamine,12-methoxydodecenoic acid, N-formyl-Met-Leu-Phe, gallamine, IAA 94, andchlorotrianisene. See U.S. Pat. No. 8,168,428 to Zon et al.

Some versions of the differentiation medium of the present inventioninclude an anti-inflammatory agent. Exemplary anti-inflammatory agentsinclude p38 MAPK inhibitors, butein, and sodium selenite, among others,which can be included at the concentrations described above.

Other components that may be included in the media of the presentinvention include serum, a glutamine supplement, a sodium pyruvatesupplement, a non-essential amino acid supplement, holo-transferrin, anda reducing agent. The serum may be fetal bovine serum, such asheat-inactivated fetal bovine serum. The serum may be included in afinal amount of about 15% v/v. The glutamine supplement, sodium pyruvatesupplement, and non-essential amino acid supplement may comprise any ofsuch supplements known in the art. An exemplary glutamine source isGlutamax™ (Life Technologies, Carlsbad, Calif.), which may be added atIX according to the manufacturer's instructions. The holo-transferrinmay be included in an amount from about 2 μg/ml to about 20,000 μg/ml,such as about 2, 50, 100, 150, 200, 150, 300, 350, 400, 450 μg/ml ormore. The reducing agent may include any reducing agent. Exemplaryreducing agents are betamercaptoethanol and dithiothreitol.

The components described above are preferably included in a minimalessential medium. Many minimal essential media are well known in theart. The minimal essential media typically include glucose, amino acids,vitamins, inorganic salts, and other basic components to supportcellular maintenance. Exemplary minimal essential media includeDulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12)(Life Technologies, Carlsbad, Calif.), Iscove's Modified Dulbecco'sMedium (IMDM) (Life Technologies, Carlsbad, Calif.). any of the variousHam's media, and alpha minimum essential medium, among others.

The differentiation medium of the present invention may include any one,all, or any combination or subcombination of the components describedabove, which are all available from commercial vendors. In certainembodiments, the differentiation medium of the present inventioncomprises a retinoic acid signaling inhibitor in combination with anyone, all, or subcombinations of BMP4, VEGF, an antioxidant, a stimulantof the prostaglandin E2 pathway, TPO, SCF, FLT-3, EPO, TGFβ1, a p38 MAPKinhibitor, a beta adrenergic receptor agonist, a cell cycle inhibitor,an RXR agonist, a chromatin remodeler, and Cripto. In certainembodiments, the differentiation medium of the present inventioncomprises BMP4 in combination with any one, all, or subcombinations of aretinoic acid signaling inhibitor, VEGF, an antioxidant, a stimulant ofthe prostaglandin E2 pathway, TPO, SCF, FLT-3, EPO, TGFβ1, a p38 MAPKinhibitor, a beta adrenergic receptor agonist, a cell cycle inhibitor,an RXR agonist, a chromatin remodeler, and Cripto. In certainembodiments, the differentiation medium of the present inventioncomprises VEGF in combination with any one, all, or subcombinations of aretinoic acid signaling inhibitor, BMP4, an antioxidant, a stimulant ofthe prostaglandin E2 pathway, TPO, SCF, FLT-3, EPO, TGFβ1, a p38 MAPKinhibitor, a beta adrenergic receptor agonist, a cell cycle inhibitor,an RXR agonist, a chromatin remodeler, and Cripto. In certainembodiments, the differentiation medium of the present inventioncomprises an antioxidant in combination with any one, all, orsubcombinations of a retinoic acid signaling inhibitor, BMP4, VEGF, anantioxidant, a stimulant of the prostaglandin E2 pathway, TPO, SCF,FLT-3, EPO, TGFβ1, a p38 MAPK inhibitor, a beta adrenergic receptoragonist, a cell cycle inhibitor, an RXR agonist, a chromatin remodeler,and Cripto. In certain embodiments, the differentiation medium of thepresent invention comprises a stimulant of the prostaglandin E2 pathwayin combination with any one, all, or subcombinations of a retinoic acidsignaling inhibitor, BMP4, VEGF, an antioxidant, TPO, SCF, FLT-3, EPO,TGFβ1, a p38 MAPK inhibitor, a beta adrenergic receptor agonist, a cellcycle inhibitor, an RXR agonist, a chromatin remodeler, and Cripto. Incertain embodiments, the differentiation medium of the present inventioncomprises TPO in combination with any one, all, or subcombinations of aretinoic acid signaling inhibitor, BMP4, VEGF, an antioxidant, astimulant of the prostaglandin E2 pathway, SCF, FLT-3, EPO, TGFβ1, a p38MAPK inhibitor, a beta adrenergic receptor agonist, a cell cycleinhibitor, an RXR agonist, a chromatin remodeler, and Cripto. In certainembodiments, the differentiation medium of the present inventioncomprises SCF in combination with any one, all, or subcombinations of aretinoic acid signaling inhibitor, BMP4, VEGF, an antioxidant, astimulant of the prostaglandin E2 pathway, TPO, FLT-3, EPO, TGFβ1, a p38MAPK inhibitor, a beta adrenergic receptor agonist, a cell cycleinhibitor, an RXR agonist, a chromatin remodeler, and Cripto. In certainembodiments, the differentiation medium of the present inventioncomprises FLT-3 in combination with any one, all, or subcombinations ofa retinoic acid signaling inhibitor, BMP4, VEGF, an antioxidant, astimulant of the prostaglandin E2 pathway, TPO, SCF, EPO, TGFβ1, a p38MAPK inhibitor, a beta adrenergic receptor agonist, a cell cycleinhibitor, an RXR agonist, a chromatin remodeler, and Cripto. In certainembodiments, the differentiation medium of the present inventioncomprises EPO in combination with any one, all, or subcombinations of aretinoic acid signaling inhibitor, BMP4, VEGF, an antioxidant, astimulant of the prostaglandin E2 pathway, TPO, SCF, FLT-3, TGFβ1, a p38MAPK inhibitor, a beta adrenergic receptor agonist, a cell cycleinhibitor, an RXR agonist, a chromatin remodeler, and Cripto. In certainembodiments, the differentiation medium of the present inventioncomprises TGFβ1 in combination with any one, all, or subcombinations ofa retinoic acid signaling inhibitor, BMP4, VEGF, an antioxidant, astimulant of the prostaglandin E2 pathway, TPO, SCF, FLT-3, EPO, a p38MAPK inhibitor, a beta adrenergic receptor agonist, a cell cycleinhibitor, an RXR agonist, a chromatin remodeler, and Cripto. In certainembodiments, the differentiation medium of the present inventioncomprises a p38 MAPK inhibitor in combination with any one, all, orsubcombinations of a retinoic acid signaling inhibitor, BMP4, VEGF, anantioxidant, a stimulant of the prostaglandin E2 pathway, TPO, SCF,FLT-3, EPO, TGFβ1, a beta adrenergic receptor agonist, a cell cycleinhibitor, an RXR agonist, a chromatin remodeler, and Cripto. In certainembodiments, the differentiation medium of the present inventioncomprises a beta adrenergic receptor agonist in combination with anyone, all, or subcombinations of a retinoic acid signaling inhibitor,BMP4, VEGF, an antioxidant, a stimulant of the prostaglandin E2 pathway,TPO, SCF, FLT-3, EPO, a p38 MAPK inhibitor, TGFβ1, a cell cycleinhibitor, an RXR agonist, a chromatin remodeler, and Cripto. In certainembodiments, the differentiation medium of the present inventioncomprises a cell cycle inhibitor in combination with any one, all, orsubcombinations of a retinoic acid signaling inhibitor, BMP4, VEGF, anantioxidant, a stimulant of the prostaglandin E2 pathway, TPO, SCF,FLT-3, EPO, a p38 MAPK inhibitor, TGFβ1, a beta adrenergic receptoragonist, an RXR agonist, a chromatin remodeler, and Cripto. In certainembodiments, the differentiation medium of the present inventioncomprises an RXR agonist in combination with any one, all, orsubcombinations of a retinoic acid signaling inhibitor, BMP4, VEGF, anantioxidant, a stimulant of the prostaglandin E2 pathway, TPO, SCF,FLT-3, EPO, a p38 MAPK inhibitor, TGFβ1, a beta adrenergic receptoragonist, a cell cycle inhibitor, a chromatin remodeler, and Cripto. Incertain embodiments, the differentiation medium of the present inventioncomprises a chromatin remodeler in combination with any one, all, orsubcombinations of a retinoic acid signaling inhibitor, BMP4, VEGF, anantioxidant, a stimulant of the prostaglandin E2 pathway, TPO, SCF,FLT-3, EPO, a p38 MAPK inhibitor, TGFβ1, a beta adrenergic receptoragonist, a cell cycle inhibitor, an RXR agonist, and Cripto. In certainembodiments, the differentiation medium of the present inventioncomprises Cripto in combination with any one, all, or subcombinations ofa retinoic acid signaling inhibitor, BMP4, VEGF, an antioxidant, astimulant of the prostaglandin E2 pathway, TPO, SCF, FLT-3, EPO, a p38MAPK inhibitor, TGFβ1, a beta adrenergic receptor agonist, a cell cycleinhibitor, an RXR agonist, and a chromatin remodeler. In theseembodiments, the retinoic acid inhibitor may include any one, all, orany subcombinations of DEAB, disulfiram (tetraethylthiuram disulfide orbis(diethylthiocarbamyoyl) disulfide), retinoic acid receptor-Oinhibitor LE135, the pan RA antagonist AGN 193109, and agonists of CYP26or other molecules that aid in RA signaling inhibition or degradation.The antioxidant may include any one, all, or any subcombinations ofsodium selenite, butein, N-acetyl cysteine, glutathione, and ascorbicacid. The chromatin remodeler may include any one, all, or anysubcombinations of valproic acid, chlamodicin, sodium buterate,mocetinostat, NVP-LAQ824, Mi-2beta, inositol, and vorinostat. Thestimulant of prostaglandin E2 pathway may include any one, all, or anysubcombinations of prostaglandin E2, PGE2, PGI2, linoleic acid,13(s)-HODE, LY171883, mead acid, eicosatrienoic acid,epoxyeicosatrienoic acid, ONO-259, Cay1039, 19(R)-hydroxy PGE2,16,16-dimethyl PGE2 p-(p-acetamidobenzamido) phenyl ester, 1l-deoxy-16,16-dimethyl PGE2,9-deoxy-9-methylene-16,16-dimethyl PGE2,9-deoxy-9-methylene PGE2, butaprost, sulprostone, PGE2 serinol amide,PGE2 methyl ester, 16-phenyl tetranor PGE2,15(S)-15-methyl PGE2,15(R)-15-methyl PGE2, BIO, 8-bromo-cAMP, forskolin, bapta-AM, fendiline,nicardipine, nifedipine, pimozide, strophanthidin, lanatoside, L-Arg,sodium nitroprusside, sodium vanadate, bradykinin, mebeverine,flurandrenolide, atenolol, pindolol, gaboxadol, kynurenic acid,hydralazine, thiabendazole, bicuculline, vesamicol, peruvoside,imipramine, chlorpropamide, 1,5-pentamethylenetetrazole,4-aminopyridine, diazoxide, benfotiamine, 12-methoxydodecenoic acid,N-formyl-Met-Leu-Phe, gallamine, IAA 94, and chlorotrianisene. The p38MAPK inhibitor may include any one, all, or any subcombinations ofLY2228820 SB 203580, BIRB 796 (Doramapimod), SB 202190, VX-702, VX-745,and PH-797804. The beta adrenergic receptor agonist may include any one,all, or any subcombinations of norepinephrine, epinephrine, salmeterol,and isoproterenol.

One exemplary version of the differentiation medium of the presentinvention includes DMEM/F12, characterized FBS, Glutamax™ (100×), sodiumpyruvate (100×), non-essential amino acids (100×), holo-transferrin,ascorbic acid, BME (1000×), PGE₂, TPO (human), SCF (human), FLT-3(human), EPO, BMP4 (human), TGFb1 (human), VEGF (human), and DEAB.Another exemplary version includes a retinoic acid signaling inhibitorsuch as DEAB and/or an RXR agonist with BMP4. Another exemplary versionincludes BMP4 in combination with norepinephrine and/or in the presenceof neural crest stem cells or their differentiation progeny. Anotherexemplary version includes one, all, or any subcombination of ascorbicacid, butein, sodium selenite, NAC (N-Acetyl Cysteine), and glutathionein combination with BMP4 and a retinoic acid signaling inhibitor such asDEAB. Another exemplary version includes an anti-inflammatory (p38inhibitor, butein, and/or sodium selenite) with BMP4 and DEAB. Otherexemplary versions are described in the Examples that follow.

In addition to the differentiation media, the invention also providesmethods of generating a second type of cell from a first type of cellwith the differentiation media described herein. The cells arepreferably from mammalian origin and more preferably from human origin.One method comprises differentiating pluripotent stem cells intoprimitive blood cells. The pluripotent stem cells may comprise ES cellsor iPS cells. The primitive blood cells may comprise parental cells ofthe blood cell lineage (e.g., CD45+, CD43+), hematopoietic progenitorcells (e.g., CD45+, CD43+, CD34+), and/or hematopoietic stem cells(e.g., CD45+, CD43+, CD34+, CD38−, CD90+, CD45RA−).

Some of the molecular pathways targeted by the compounds and theireffects on developmental processes are conserved across species fromzebrafish to frog to mammals and as such may have applications wherecellular expansion of one type of cell in one species can be used and isideal for production of biologically useful (including therapeuticallyuseful) molecules or cells for similar or different species.

An exemplary method is shown in FIGS. 2A and B, wherein pluripotent stemcells (ES cells or iPS cells) are differentiated through mesoderm,lateral plate mesoderm, hematopoietic mesoderm, and hemogenicendothelium to form hematopoietic stem cells or hematopoietic progenitorcells. An advantage of the present invention is that a singledifferentiation medium as described herein can be used indifferentiating pluripotent stem cells all the way to hematopoietic stemcells or hematopoietic progenitor cells without changing componentsthereof. Furthermore the invention described herein takes advantage ofthe broad function of RA inhibition to slow differentiation andproliferation, particularly of sensitive cell types such as HSCs, andalso takes advantage of the broad role of reduced RA signaling indirected differentiation of cell types that are precursors of blood inthe majority of developmental stages during embryonic development. Thedifferentiation media can also be used in any substep betweendifferentiating from pluripotent stem cells to hematopoietic stem cellsor hematopoietic progenitor cells, such as differentiating mesoderm tolateral plate mesoderm, differentiating lateral plate mesoderm tohematopoietic mesoderm, differentiating hematopoietic mesoderm to aorticendothelium, etc.

As shown in FIGS. 2A and 2B, the exemplary method of differentiatingpluripotent stem cells to hematopoietic stem cells or hematopoieticprogenitor cells is carried out over a period of about 16 days or more.Human pluripotent stem cells are expanded in standard expansionconditions until large, undifferentiated colonies are present (comparedto a size normally observed before a standard split). The colonies aremaintained daily with standard stem cell expansion procedures.Differentiated colonies are marked and removed. Remaining colonies arethen transferred to a dish (e.g., 10-cm dish) for EB suspension culture.Media used for this first step may be about 10 ml of EB media (DMEM/F12+15% heat-inactivated fetal bovine serum (hiFBS), 1× non-essential aminoacids (NEAA), 1× Glutamax). This day of the experiment is referred to asDay 0. The following day (Day 1), the EBs are brought up from thesuspension dish and washed in a fitting conical tube. They are returnedto their dish with 10 ml fresh media composed of about 50% EB medium and50% differentiation medium. The following day (Day 2), the procedure isrepeated but with 100% differentiation medium. Thereafter, 50% of themedia is replaced for each dish with fresh media every second day. AtDay 8, the EBs are transferred from their suspension dish to Matrigel™(BD Biosciences, San Diego, Calif., www.bdbiosciences.com) coated wellson a 6-well plate. Cystic EBs are a good sign but no guarantee for goodEB quality in regard to final blood output. When the EBs are plated intothe 6 wells, their total medium volume per well (2-3 ml) is preferably50% old media and 50% fresh differentiation medium. Additionaldifferentiation medium is added to each well (preferably 1 ml ofadditional fresh media) every second day until the endpoint of theprotocol. The EBs will settle down on the surface of the wells andinitiate rapid spread of endothelial-like cells into their surroundings.At Day 12, one can start to observe small round cells budding fromcertain endothelial spreads. These cells are likely to be the firstemerging cells of hematopoietic potential. The cultures are preferablyallowed to keep going until Day 16. At the last day of the protocol(preferably Day 16), the cultures are singularized, preferably by usingTrypLE (Life Technologies, Carlsbad, Calif.), mechanical shearing, andcell filtration (30 micron). Once harvested, the cells are ready foranalysis (FACS, CFU-assay, transplantation, or other methods of analysisor treatment). This exemplary method is provided only as an example andcan be modified in a number of ways.

As described above, large, dense embryoid bodies (EBs) are preferablyformed from the pluripotent stem cells prior to exposing the cells tothe differentiation medium. Accordingly, the EBs are preferably formedfor a period of at least about 6.5, about 7, about 7.5, about 8, or more10 days before exposing the EBs to the differentiation medium. The EBsinitially exposed to the differentiation medium preferably are betweenabout 0.5 mm and about 3 mm in diameter, such as between about 1 mm and2 mm in diameter. The EBs initially exposed to the differentiationmedium preferably are cystic, having “bubble”-like masses of cellsprotruding about the EB periphery. Although, it is initially preferredto expose EBs to the differentiation medium as described above, theinvention also can be performed by initially exposing thedifferentiation medium to dispersed cells, dissociated cells, cellcolonies, non-cystic EBs, or EBs having a diameter smaller than theranges specified above.

The 50% EB medium and 50% differentiation medium used to culture thecells (e.g., EBs, etc.) at Day 0 may be called a “mixed medium,” asopposed to a pure non-differentiation medium (e.g., EB medium) or a puredifferentiation medium. The cells (e.g., EBs, etc.) may be cultured inthe mixed medium for a period other than about 24 hours, such as aperiod ranging from about 12 to about 48 hours.

After replacing the mixed medium, it is preferred that freshdifferentiation medium is added only to existing differentiation mediumsuch that at no point after replacing the mixed medium is existingmedium entirely removed from the cells. “Fresh medium” as used hereinrefers to medium that has not been exposed to cells intended to bedifferentiated. “Existing medium” as used herein refers to medium thathas been exposed to cells being differentiated or intended to bedifferentiated for a period of at least about 15 minutes, 30 minutes, 1hour, 2 hours, 6 hours, 12 hours, 18 hours, or 24 hours. The partialexchange of medium and addition of medium help to dilute toxicbyproducts and replenish nutrients while maintaining possible unknownfactors made by the cells that aid in the differentiation process.Although it is preferred that fresh differentiation medium is added onlyto existing differentiation medium after replacing the mixed medium, theinvention also can be performed by entirely replacing thedifferentiation medium after replacing the mixed medium.

It is preferred that the cells are exposed to the differentiation mediumfor a period of at least about 5 days prior to plating the cells. Thecells may be plated on any adherent surface. The surface may be a2-dimensional surface or a 3-dimensional surface. The surface may becoated with one, all, or any combination of the components selected fromthe group consisting of BD Matrigel™ (BD Biosciences, San Diego, Calif.,www.bdbiosciences.com) Geltrex® (Life Technologies, Carlsbad, Calif.),Laminin and/or its derivatives, Entactin (and its derivatives),vitronectin or one or more fragments or peptides of vitronectin (e.g.,Vitronectin XF™ from Primorigen Biosciences, Madison, Wis.,www.primorigen.com), collagen (one or more fractions I-IV), or syntheticor RGD-containing peptide coated surfaces (e.g., Synthemax® fromCorning, Tewksbury, Mass.).

It is preferred that the cells are plated in the absence of feedercells. However, the invention also may be performed by plating in thepresence of feeder cells.

After plating, it is preferred that fresh volumes of the differentiationmedium are added only to existing volumes of the differentiation mediumwithout removing any of the existing volume of the differentiationmedium. However, the invention also may be performed by completelyreplacing the existing volume of differentiation medium with a freshvolume of differentiation medium or by removing only some of theexisting volume of differentiation medium.

In some versions of the invention, the cells are cultured in thepresence of neural crest stem cells in addition to or as an alternativeto exposing the cells to the differentiation medium. Neural crest stemcells and their progeny cells appear to provide multiple additionalfactors promoting the generation of HSCs (e.g., via cell contact,secreted factors, etc).

The method of differentiating cells described above may additionallycomprise further differentiating the generated primitive hematopoieticcells into more highly differentiated blood cells. The more highlydifferentiated blood cells may include any differentiated blood cellsdescribed herein. Exemplary differentiated blood cells include any ofthe various types of myeloid cells and any of the various types oflymphoid cells. The primitive hematopoietic cells may be furtherdifferentiated in vitro or in vivo. In vitro methods of differentiatingprimitive hematopoietic blood cells are well known in the art. See,e.g., Charoudeh et al. Blood. 2010 July 15; 116(2):183-92 for methodsfor differentiating primitive hematopoietic blood cells intolymphocytes. In addition, any methods currently used to differentiatecord blood cells can be used to further differentiate the primitivehematopoietic blood cells generated as described herein. In vivo methodsof differentiating the primitive hematopoietic blood cells includeadministering the cells into the bloodstream of an animal, as occursduring engraftment with cord blood cells. The administered cells willdifferentiate into various forms within the animal.

Accordingly, and as described in further detail below, the inventionalso provides methods of engrafting a blood cell in an animal. Theanimal is preferably a human. The methods generally compriseadministering a primitive hematopoietic stem cell generated by themethods described herein or a cell differentiated therefrom to theanimal. Methods of engraftment are described in U.S. Pat. No. 8,168,428to Zon et al. In some versions, the animal is a candidate for bonemarrow or stem cell transplantation, or an animal that has received bonemarrow ablating chemotherapy or irradiation therapy.

The differentiation media, the differentiation methods, and the cellsgenerated therewith may be used in any of a number of embodiments.

One embodiment comprises differentiating pluripotent stem cells (ES oriPS) to a definitive HSC phenotype (CD45+, CD43+, CD34+, CD38−, CD90+,CD45RA−) with lymphoid and myeloid differentiation capacity for researchand therapeutic purposes.

Another embodiment comprises using such cells for in vitro studies ofhematological diseases. This may comprise further differentiating theHSC phenotype cells as produced herein, themselves differentiated frompluripotent stem cells, into various terminal blood cells and used tobuild assays for the disease or to identify drugs and medicines to treatsuch diseases. For example, in certain osteoporosis diseases, iPS cellsgenerated from a patient can be used to generate HSCs and then principleblood components therefrom. See, e.g., Example 5. One can then identifyreduced macrophage function as a surrogate for the disease. One canfurther devise treatments for the disease using the cells producedherein or derived from the cells produced herein to identify genetherapy based treatments to restore macrophage function and amelioratethe manifestations of the disease.

In another embodiment, in vitro studies of developmental hematopoiesiscan be performed by investigating the precursors to the HSCs generatedwith the methods described herein. Such precursors can be used toidentify developmental factors that influence the cells toward aparticular cell type or other condition of interest. For example, onecould explore potential molecules and other treatments to studyposterior lateral plate mesoderm formation by looking at markers ofFOXC1, FOXF1, SALL1, or others, as described herein.

In another embodiment, in vitro studies of hematopoietic stressresponses can be performed by focusing on the most primitivehematopoietic cells resulting from the invention described herein. Forexample, using p38, a molecule that reduces NFKb signaling andinflammation responses, can increase HSC output. See Example 1 for anexample of a screen that can be used in such a study.

In another embodiment, in vivo zenograft studies can be performed usingcells produced from the methods described herein to better evaluatedisease cells and genetically corrected disease cells. Such studies canalso be performed to assess compounds applied to the disease cells or tothe animal itself for their ability to correct the disease phenotypewhen the animal is administered the HSCs or progenitors produced asdescribed herein.

In another embodiment, HSCs produced in vitro from pluripotent stemcells using the methods described herein can also be used to evaluate,screen, and identify additional novel factors to generate definitiveHSCs with clinically relevant transplantation potential.

In another embodiment, patients with hematological disease, or diseasewhere blood cell transfusion can alleviate, reduce, or abrogatesymptoms, can be transfused with the HSCs or progenitors produced asdescribed herein. For example, patient-derived iPS cells from a patientsuffering from sickle cell anemia can be used to produce HSCs andprogenitors using the invention described herein and can be furtherdifferentiated into erythrocytes (following genetic correction at somepoint whether prior to iPS generation, at the iPS stage, or downstreamof the iPS) to treat the sickle cell anemia.

In another embodiment, HSCs or progenitors produced according to themethods described herein can be used to produce granulocytes andmacrophages for therapeutic purposes. For example, HSCs and progenitorsproduced using the invention described herein can be differentiatedfurther into neutrophils, eosinophils, monoblasts, myeloblasts,macrophages, or megakaryocytes (for platelet production). These terminalcells can be transplanted into a patient or otherwise used fortherapeutic purposes. Alternatively, patient iPS-derived HSCs producedaccording to the methods described herein can be transplanted (followinggenetic correction at some point whether prior to iPS generation, at theiPS stage, or downstream of the iPS) into chronic granulomatous diseasepatients. Alternatively, myeloid progenitors or their progeny (maturemacrophages and granulocytes) derived from progenitors and HSCsdifferentiated from pluripotent stem cells using the invention describedherein can be transfused into these patients to treat the disease.

In another embodiment, HSCs and progenitors produced from pluripotentstem cells using the invention described herein can be furtherdifferentiated into B, T, and NK (lymphoid cells), and these terminallydifferentiated cells can then be transplanted into a human in order totreat symptoms of a disease resulting from abnormalities in their invivo lymphoid cell production systems.

In another embodiment, T-cells generated from progenitors or HSCsdifferentiated from pluripotent stem cells according to the inventiondescribed herein can be engineered at either the iPS cell stage or laterin differentiation to have anti-tumor activity by selectively targetingthe tumor for destruction by immune cells initiated by T-cell anti-tumorresponse. T-cells can be engineered to specifically target epitopesuniquely expressed on tumor cells as reported in Morgan et al., HumanGene Therapy. October 2012, 23(10): 1043-1053.

In another embodiment, HSCs and progenitors produced frompatient-derived pluripotent stem cells according to the inventiondescribed herein can be used directly for drug screens to identifypromising drug candidates for treating hematological diseases.Additionally, HSCs and progenitors produced from pluripotent stem cellsaccording to the invention described herein can be furtherdifferentiated using many different methods into terminal blood celltypes. The terminally differentiated cells can then be used to constructand conduct drug screens to identify promising drug candidates fortreatment of the various hematological diseases. For example, largenumbers of helper t1 (Th1) cells and helper t2 (Th2) cells can bederived from progenitors and HSCs produced from pluripotent stem cellsaccording to the invention described herein. The cells can then beimmobilized in a high throughput surface, such as a 96-well or 384-wellmicrotiter plate coated with a supportive coating and immersed in amedium supporting the Th1 and Th 2 cells. The Th1 and Th2 cells can thenbe used to screen drug candidates for the ability to modulate the immuneresponses facilitated or generated from Th1 and Th2 cells. For example,libraries of compounds could be added to the wells containing the Th1and/or Th2 cells and after an appropriate incubation time, any number ofoutputs could be measured using fluorescence, chemiluminescence, orother enzymatic or other methods to record the output and thus identifythe effect of the compound on Th1 and/or Th2 cell activity. A furtherexample would be using the Th1 and Th2 cells derived as described hereinto screen derivatives of prostaglandins for their ability to effectproduction of cytokines such as interleukin-2 (IL-2), IL-4, IL-5, andInterferon Gamma and others.

There are approximately 120 hematological disorders, all of which couldbenefit from HSCs and progenitors differentiated from pluripotent stemcells according to the invention described herein. These progenitors andHSCs differentiated from pluripotent stem cells according to theinvention described herein could be used directly to treat thesediseases following proper conditioning of the patient or furtherdifferentiated into the terminal cell directly affected in theparticular disease and delivered to the patient who had a deficiency ordefect in a particular terminal cell type. For example, X-Linked severecombined immunodeficiency disease patients are missing entire lymphoidlineages (B cells, T cells, NK cells). Transplanting these patients withprogenitors and/or HSCs differentiated from pluripotent stem cellsaccording to the invention described herein or with terminal B cells, Tcells, NK cells further differentiated from progenitors and HSCsdifferentiated from pluripotent stem cells according to the inventiondescribed herein with the corrected gene could restore lineage functionto these patients. For example, transplanting the patients sufferingfrom diamond blackfan anemia (red cell deficiency) with progenitorsand/or HSCs differentiated from pluripotent stem cells according to theinvention described herein, with erythroid progenitors derived fromprogenitors and HSCs differentiated from pluripotent stem cellsaccording to the invention described herein, or with terminal red bloodcells (erythrocytes) cells further differentiated from progenitors andHSCs differentiated from pluripotent stem cells according to theinvention described herein could provide a significant treatment forthese patients.

In another embodiment, HSCs, progenitors or mature blood cells producedfrom pluripotent stem cells according to the invention described hereincan be used to provide therapeutic factors generated within the cellsfollowing genetic manipulation of the cells for treatingnon-hematological diseases. For example, blood cells generated frompluripotent stem cells can be modified to overexpress clotting factorgenes (Factor IX) and transplanted/transfused into hemophilia patients.These clotting factors normally are produced in the liver but could beproduced in blood cells allowing for systemic delivery of therapeuticproteins and factors.

The elements and method steps described herein can be used in anycombination whether explicitly described or not.

All combinations of method steps as used herein can be performed in anyorder, unless otherwise specified or clearly implied to the contrary bythe context in which the referenced combination is made.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the content clearly dictates otherwise.

Numerical ranges as used herein are intended to include every number andsubset of numbers contained within that range, whether specificallydisclosed or not. Further, these numerical ranges should be construed asproviding support for a claim directed to any number or subset ofnumbers in that range. For example, a disclosure of from 1 to 10 shouldbe construed as supporting a range of from 2 to 8, from 3 to 7, from 5to 6, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

All patents, patent publications, and peer-reviewed publications (i.e.,“references”) cited herein are expressly incorporated by reference tothe same extent as if each individual reference were specifically andindividually indicated as being incorporated by reference. In case ofconflict between the present disclosure and the incorporated references,the present disclosure controls.

It is understood that the invention is not confined to the particularconstruction and arrangement of parts herein illustrated and described,but embraces such modified forms thereof as come within the scope of theclaims.

EXAMPLES Materials and Methods Overview

Human ES cell lines and iPS cell lines were differentiated towards theblood lineage using a modified version of a previously describedprotocol (Woods et al. Stem Cells, 2011, 29(7):1158-1164). Themodifications, which improved the efficiency of blood cell output, aredescribed as follows.

For embryoid body (EB) formation, pluripotent stem cell colonies werecultured an additional 24 hours to increase colony cell density. Duringthe EB formation stage following pluripotent stem cell colony peeling,the suspension culture was extended to 8 days with a 1.5-fold increasein media volume. A low-adherence, round 10-cm dish was used in place oflow-adherence T25 flasks. The round dish prevents the accumulation ofEBs in corners and reduces EB aggregation that reduces blood celloutput. Together, these modifications allow for a 3-fold increase numberof EBs generated per square cm.

For the adherence plating of the EBs for endothelial and hematopoieticcell expansion, the murine OP9 feeder layer was replaced with Matrigel™(BD Biosciences, San Diego, Calif., www.bdbiosciences.com) orVitronectin XF® (Primorigen Biosciences, Madison, Wis.,www.primorigen.com), both of which increased hematopoietic cell outputand increased primitive cell output with an HSC phenotype.Differentiation Medium 1 (defined below) was used continuouslythroughout the protocol and was not changed to a hematopoietic expansionmedia during the last week. Differentiation Medium 1 was added everysecond day to the adherent EB wells and none was removed to reducedilution of secreted factors from the cultured cells. The duration ofthe protocol was reduced to a total of 16 days by removing the final 5days of the published protocol.

DEAB was used throughout the differentiation protocol at variousconcentrations. DEAB was found to maintain cells with an HSC phenotypeby increasing the numbers of cells with a capacity for both lymphoid andmyeloid differentiation capacity, to increase mesodermal precursors ofblood during the differentiation process, and to block endoderm andcardiac mesoderm cell lineage specification. The long duration of theprotocol and the ability to generate both lymphoid and myeloid cells,including erythrocytes with beta hemoglobin, indicate that the protocolis capable of generating definitive hematopoietic cells.

Additional factors intended to influence mesoderm cell expansion anddifferentiation, hemogenic endothelial cell propagation, andhematopoietic cell expansion (including primitive and definitivehematopoietic), self-renewal, and maintenance were applied to theDEAB-containing Differentiation Medium 1 medium individually at varyingconcentrations and times during the differentiation protocol. Resultswere analyzed to determine additional molecules that increased furtherthe yield of progenitors and phenotypic stem cells in ourdifferentiation system.

The resulting protocol is described in detail as follows.

Pluripotent Stem Cell Validation and Culture

Human ES cell lines and iPS cell lines that were karyotypically normaland shown to be pluripotent by in vivo teratoma histological assays andpolymerase chain reaction (PCR) were obtained. The pluripotent celllines were cultured and expanded using either Matrigel™ (BD Biosciences,San Diego, Calif.) or murine embryonic fibroblast feeder cells in stemcell media (mTeSR1 or DMEM/F12 media, respectively) containing FGFb tomaintain pluripotency.

Embryoid Body Formation

Embryoid bodies (EBs) were generated from ES cells or iPS cells grown onmouse embryonic fibroblasts or Matrigel-coated 10-cm dishes for 8 days,so that colonies appeared large and dense. Colonies were separated fromthe plate with 4 ml of dispase (0.5 mg/ml, Life Technologies, Carlsbad,Calif., www.lifetechnologies.com) for 30-45 minutes. Colonies werecollected in EB medium (Iscove's modified Dulbecco's medium supplementedwith 15% fetal bovine serum (FBS) (Thermo Scientific Hyclone, Rockford,Ill., www.thermoscientific.com), 1% nonessential amino acids (LifeTechnologies, Carlsbad Calif.), and 1% GlutaMax (Life Technologies,Carlsbad Calif.), allowed to settle at the bottom of a 15-ml conicaltube, rinsed twice with EB medium, and placed in a non-adherent rounddish in EB medium overnight.

Mesoderm Generation with Posterior Lateral Plate Mesoderm Specification

Early stage EB colonies (at 24 hours) were then cultured inmesoderm-specifying Differentiation Medium 1 (described below) for atotal of 8 days with partial medium changes made every other day. Cellswere harvested at day 0, 4 and 8 for RT-PCR analysis of marker genes ofmesoderm lineage development.

Differentiation Medium 1 is composed of Dulbecco's modified Eaglemedium/F12 supplemented with batch selected FBS 15%, 10 ng/ml bonemorphogenetic protein 4 (BMP4), 5 ng/ml transforming growth factor beta1 (TGFb1), 1 ng/ml vascular endothelial growth factor (VEGF), 20 ng/mlthrombopoietin (TPO), 20 ng/ml erythropoietin (EPO), 20 ng/ml stem cellfactor (SCF), 20 ng/ml FMS-like tyrosine kinase 3 ligand (FLT3L), 200μg/ml holotransferrin, 2 μM prostaglandin E2 (PGE2), 50 μg/ml ascorbicacid, and 10 μM DEAB. Differentiation Medium 1 includes DEAB except whenexplicitly indicated otherwise. The components in Differentiation Medium1 were obtained from commercial vendors.

EB Plating on Extra Cellular Matrix for Definitive Hematopoietic CellGeneration and Expansion

Whole, day 8 EBs were then plated on growth factor reduced Matrigel™ (BDBiosciences)-coated six-well plates. The mesoderm-specifying medium(Differentiation Medium 1) was added every other day until day 16(termination of the blood generation protocol) without removal of theconditioned media.

Analysis by Flow Cytometry

Cells from the differentiation cultures were collected at time pointsindicated and washed in phosphate-buffered saline (PBS) supplementedwith 2% FBS. Adherent cells were individualized using TrypLE (LifeTechnologies, Carlsbad Calif.), passed through a 27.5-gauge needle andfiltered through a 40-70 μm cell strainer (BD Falcon, San Diego, Calif.,www.bdbiosciences.com). Cells were treated with 7-aminoactinomycin D(7AAD) before analysis, and positive cells were gated out of results.Cells were stained with fluorescence-conjugated antibodies for thehematopoietic stem and progenitor cell markers CD45, CD43, CD34, CD38,CD90, CD45RA. Cells were analyzed on a FACS Canto (BD, Franklin Lakes,N.J., www.bd.com).

Hematopoietic Progenitor Cell Differentiation Assays

The differentiation cultures were trypsinized at the indicated timepoints and single-cell suspensions were generated followingtrypsinization. For the Colony Forming Unit (CFU) assays using methylcellulose cultures, 25% of the cells were plated in 2 ml of MethoCultH4435 (STEMCELL Technologies, Vancouver, BC) in six-well plates.Alternatively, sorted CD45+, CD43+, CD34+, 7AAD− cells were sorted andplated at 500 cells per 2 ml Methocult media. Cells were incubated for14 days in a humidified incubator at 37° C. with 5% CO₂ and evaluatedfor colonies by bright-field microscopy (Zeiss (Oberkochen, Germany,www.zeiss.com) Axiovert 200 with a Zeiss Axiocam). Cells wereresuspended in PBS supplemented with 2% FBS. For the lymphoid myeloiddifferentiation assays, either CD45+, CD43+, CD34+, 7AAD− hematopoieticprogenitors, or CD45+, CD43+, CD34+, CD38−, CD90+, CD45RA− phenotypicHSCs were plated at 20-40 cells per well onto OP9 and OP9delta feederlines for B-cell and T-cell differentiation, respectively, as previouslydescribed (Nozad Charoudeh H, Tang Y, Cheng M, Cilio C M, Jacobsen S E,Sitnicka E., Identification of an NK/T cell-restricted progenitor inadult bone marrow contributing to bone marrow- and thymic-dependent NKcells., Blood. 2010 July 15; 116(2):183-92). NK cell differentiation aswell as myeloid cell differentiation was also detected in thisdifferentiation system. FACS analysis was performed 4-6 weeks afterplating for markers of lymphoid cell differentiation with the markersindicated. Similar cell sortings were performed for single cell Terasakiplate assays of myeloid differentiation and expansion using StemSpanmedia (STEMCELL Technologies, Vancouver, BC) supplemented with TPO, SCF,FLT3, IL3, GM-CSF. Colony size was estimated by microscopy.

Mesoderm Gene Expression Analysis

RNA from the pluripotent stem cell differentiation cultures washarvested at multiple time points corresponding to stages of developmentfrom pluripotent stem cells to hematopoietic lineages during in vitroculture. Cells were lysed using Trizol (Life Technologies, CarlsbadCalif.), and RNA was isolated according to the manufacturer'sspecifications. A cDNA library was reverse transcribed by the HighCapacity RNA-to-cDNA Kit from Applied Biosystems (Life Technologies,Carlsbad, Calif.). PCR products were amplified for 40 cycles usingprimers designed for the gene of interest.

Quantitative Analysis of Cell Numbers and Statistical Analyses

Cells were counted using the FACS cell counter feature (BD) or bycytometer using a bright-field microscope to obtain viable cell numbers.The total numbers of cells of each subpopulation were calculated bymultiplying the number of cells by the proportion of cells of thatlineage as determined by flow cytometry. Statistical analyses of datapoints and error bars of this study show mean and standard deviation,with statistical significance assessed by the Student's t-test.

Pluripotent Stem Cell Derived Hematopoietic Cell Transplantation Assays

Single-cell suspensions of the pluripotent stem cell differentiationcultures described herein were injected into sublethally irradiated (325rad) NOD.Cg-Prkdc^(scid) Il2rg^(tmlWjl)/SzJ mice (JAX, Bar Harbor, Me.,www.jax.org) via tail vein injections, intrafemoral injection, orcardiac injection. Peripheral blood was harvested at times indicated viatail vein or retro-orbital eye bleed or cardiac puncture on anesthetizedmice. Bone marrow and spleen cells were harvested at the terminal stageof the transplant experiment. Bone marrow cells were isolated via pestlemortar crushing of bone followed by rinsing and 70 micron filtering.Cells were analyzed by FACS, CFU assay, or transplanted serially intosecondary and tertiary mice. FACS analysis for the human panhematopoietic marker CD45, progenitor marker CD34, myeloid markersCD11b, CD15, CD33 and lymphoid markers CD19, CD56 and CD132, anderythroid markers CD235. When erythrocytes were not being analyzed, thered cells were lysed using ammonium chloride solution (0.8% NH₄Cl with0.1 mM EDTA) (STEMCELL Technologies, Vancouver, BC), spun, and stainedwith antibodies against the above-mentioned markers. All animalexperiments were conducted in accordance with Institutional Animalethical use protocols.

Example 1 Small Molecule Screen

A small molecule screen was performed to identify compounds that improvethe output of hematopoietic progenitor cells (HPCs; CD45+, CD43+, CD34+)and phenotypic hematopoietic stem cells (HSCs; CD45+, CD43+, CD34+,CD38−, CD90+, CD45RA−) differentiated from pluripotent stem cells.Molecules targeting pathways known or suspected of being important inhematopoiesis were screened. The screened molecules included garcinol(10 μM), a histone acetyltransferases (HATs) inhibitor; pifithrinA (10μM), a p53 tumor suppressor protein inhibitor, to minimize DNA damagefrom oxidative stress; CHIR99021 (1 μM), a GSK3 inhibitor, to activatethe Wnt pathway for enhancing specification of lateral plate mesoderm toposterior lateral plate mesoderm; SRT1720 (1 μM), an activator orenhancer of SIRT1 enzymatic efficiency; deferoxamine (1 μM), an inducerof hypoxia-like responses in cells through induction/activation ofHIF-1a; diethylaminobenzaldehyde (DEAB; 10 μM), a retinoic acidsynthesis or accumulation inhibitor, and rapamycin (0.1 μM), aninhibitor or silencer of the mTOR pathway. Each of the above moleculeswas added to a base medium essentially as published in Woods et al. StemCells, 2011, 29(7):1158-1164 at the above-described final concentrationsand used to differentiate ES and iPS cells. Cells were analyzed usingfluorescence activated cell sorting (FACS; FIGS. 3A, 3B, and 3C) andCFU-GEMM assays (FIG. 3D). As shown in FIGS. 3A-D, both DEAB andrapamycin increased the output of total blood cells (CD45+, CD43+; FIG.3C), HSCs (FIGS. 3A and B), and myeloid progenitors (FIG. 3D).

Example 2 Effects of DEAB

From Example 1, DEAB was selected to move forward into an experimentfurther establishing the phenotypes of blood cells emerging from thedifferentiation in order to identify at what stage of differentiationDEAB may exert its effects. Pluripotent stem cells (iPS and ES) weregrown and induced to produce embryoid bodies on Day 8 as described. TheEBs were then exposed to Differentiation Medium 1 for a total of 8 dayswith partial medium changes made every other day as described. Cellswere harvested on Day 16 and washed in phosphate-buffered saline (PBS)supplemented with 2% FBS. Adherent cells were individualized usingTrypLE (Life Technologies, Carlsbad Calif.), passed through a 27.5-gaugeneedle and filtered through a 40-70 μm cell strainer (BD Falcon, SanDiego, Calif., www.bdbiosciences.com). Cells were treated with7-aminoactinomycin D before analysis and positive cells were gated outof results. Cells were stained with fluorescence conjugated antibodiesfor the hematopoietic stem and progenitor cell markers CD45, CD43, CD34,CD38, CD90, CD45RA. Cells were analyzed on an FACS Canto (BD, FranklinLakes, N.J., www.bd.com).

Results are presented in FIG. 4A (total cell counts), FIG. 4B (foldchange in total cell counts), FIG. 3C (percentage of harvested cellsexpressing these markers), and FIG. 4D (fold change in total cellpercentages expressing these markers). Taken together, these resultsshow that presence of DEAB at 10 uM results in modestly higher values oftotal viable cells, blood cells (CD45+, CD43+), hematopoietic progenitorcells (CD45+, CD43+, CD34+), and substantial improvement in thepopulation of cells exhibiting a hematopoietic stem cell phenotype(CD45+, CD43+, CD34+, CD38−, CD90+, CD45RA−). Collectively these datashow that DEAB when used in this differentiation system exerts an effecton increasing the generation of the most primitive cells (FIGS. 4A and4C), with generation of the phenotypic stem cell phenotype increasingover 3-fold (FIGS. 4B and 4D).

Example 3 CFU Results with DEAB

For the Colony Forming Unit (CFU) assays using methyl cellulosecultures, 25% of the cells harvested for Example 2 were plated into in 2ml of MethoCult H4435 (STEMCELL Technologies, Vancouver, BC) in six-wellplates. Alternatively, sorted CD45+, CD43+, CD34+, 7AAD− cells weresorted and plated at 500 cells per 2 ml MethoCult media. Cells wereincubated for 14 days in a humidified incubator at 37° C. with 5% CO₂and evaluated for colonies by bright-field microscopy (Zeiss,Oberkochen, Germany, www.zeiss.com) Axiovert 200 with a Zeiss Axiocam).

The results of this experiment are shown in FIG. 5. The data show that10 uM DEAB enables measurable levels of all four major CFU assay types(CFU-G, CFU-M, CFU-GEMM, BFU-E to be obtained and that the total numberof colonies counted in the assay more than double the number of coloniesobtained from identical assay when using cells not differentiated in thepresence of DEAB (DMSO control).

Example 4 Gene Expression Results

Gene expression studies were carried out to determine expression of keygenes for the major divergences in the pathway from iPS to HSC. Briefly,RNA from the differentiation cultures was harvested at multiple timepoints (Day 0, 4, 8, 12, 15), corresponding to stages of developmentduring in vitro culture and differentiation of pluripotent stem cells tohematopoietic lineages using Differentiation Medium 1. Cells were lysedusing Trizol (Life Technologies, Carlsbad Calif.) and RNA was isolatedaccording to manufacturer's specifications. A cDNA library was reversetranscribed using the RNA-to-cDNA Kit from Applied Biosystems (LifeTechnologies, Carlsbad, Calif.). PCR products were amplified usingappropriate primers designed for the genes of interest for 40 cycles.

To understand how RA-signaling inhibition with DEAB may lead toincreased output of the most primitive blood cells (phenotypic HSCs:CD45+, CD43+, CD34+, CD38−, CD90+, CD45RA−) from pluripotent stem cellswithout an increase in the total blood (mature cells and progenitors),we performed quantitative gene expression analyses for key developmentmarkers at various time points during the differentiation protocol. Asthe developmental marker genes we surveyed are not known to be directtargets of RA signaling, we can expect the marker gene expression levelsto correlate to the cell numbers in culture.

The expression analyses identified differential expression in genesranging from early germ layer specification (PAX6, Brachyury, MIXL1,SOX17), mesodermal sub specifications (FOXF1, FOXC1, NKX2.5), hemogenicendothelial specifications (APLNR, PDGFRA (low), FLK1), andhematopoietic specifications (RUNX1). The first days of thedifferentiation involve the formation of embryoid bodies in a mesodermbiasing media (primarily due to BMP4), we saw that RA signalinginhibition by DEAB significantly increased overall expression of themesoderm marker, Brachyury (FIG. 6B), slightly increased (20%)expression of the primitive streak mesoderm marker MIXL1 (FIG. 6C),significantly reduced expression of the endoderm specific marker SOX17(FIG. 6D), and the early ectoderm and neuroectoderm marker PAX6 showedreduced expression at early time point with the inhibitor but this levellater equalized (FIG. 6A). These results suggest that RA inhibition byDEAB during germ layer specification of differentiating pluripotent stemcells increased mesoderm lineage specification at the expense ofendoderm, and had limited effect on ectodermal lineage.

Within the mesoderm lineage we then quantified the expression of markersof the paraxial (FOXC1) and lateral plate (FOXF1, hematopoietic andcardiac precursor) mesoderm. As expected both these markers showelevated levels (FIGS. 6F and 6E, respectively) at early time points incomparison to the DMSO control, likely as a result of the increasedmesodermal specification from the inhibitor earlier in the protocol.However the paraxial mesoderm marker levels FOXC1 became significantlyreduced over time with the RA inhibitor DEAB compared to the DMSOcontrol (FIG. 6F, Days 6-12), despite the initial increase in mesodermspecification, suggesting further specification of lateral platemesoderm. The lateral plate mesoderm marker levels of FOXF1 in the DMSOcontrol eventually increased to levels comparable to the RA inhibitorDEAB (FIG. 6E, Day 8 onward). This is likely the result of anteriorlateral plate mesoderm formation (precursors of the heart). We saw adramatic upregulation of the cardiogenic mesoderm marker NKX2.5 in theDMSO control at this time point (FIG. 6G, Day 12 onward). The reducedexpression of the cardiogenic lateral plate mesoderm marker NKX2.5 usingthe RA inhibitor DEAB is in agreement with the significantly reducednumbers of beating cardiomyocytes colonies seen in our cultures usingthe RA inhibitor DEAB (data not shown).

Together these data suggest that using the RA inhibitor DEAB in ourpluripotent stem cell differentiation system recapitulates the knownembryonic developmental processes through mesoderm specification towardshemogenic endothelium.

Because the markers of lateral plate mesoderm were elevated using the RAinhibitor DEAB, we then looked at two newly identified markersspecifying mesodermal cells with hematopoietic potential, APLNR andPDGFRA. APLNR has been shown to be upregulated in mesodermal cells withdefinitive or primitive blood potential (Choi et al. Cell Rep. 2012,2(3):553-67)). PDGFRA has been shown to be upregulated in mesodermalcells with mostly primitive blood potential and hemangioblast activity(Choi et al. Cell Rep. 2012, 2(3):553-67)). In definitive typehematopoietic precursors, namely hemogenic endothelium, APLNR isexpressed highly, however PDGFRA is downregulated. APLNR and PDGFRA showthis expression pattern at Day 12 in the DEAB suggesting definitivehematopoietic cell potential (FIGS. 6H and 6J, respectively). This wassubsequently confirmed by the increased expression of RUNX1 (adefinitive hematopoietic cell marker) (FIG. 6I) in the later stages ofthe differentiation (Day 12-15) compared to the DMSO control.

Interestingly, in the presence of the RA inhibitor DEAB, the marker geneFLK1 which is an endothelial cell marker but also a broad mesodermmarker (including of mesoderm with cardiac potential) (Ema et al.,“Deletion of the selection cassette, but not cis-acting elements, intargeted Flk1-lacZ allele reveals Flk1 expression in multipotentmesodermal progenitors,” Blood, 2006, 107:111-117), showed high levelsat the early time point (Day 4), when primitive hematopoietic cellgeneration is possible, and showed similar expression compared to DMSOin the intermediate time point (Day 8), when mesoderm cells werecommitting to the cardiac mesoderm in the DMSO. At later time points(Days 12-15), however, FLK expression increased in the presence of DEAB,indicating an increase endothelial cell number that agrees with theincreased blood potential seen by FACS and definitive hematopoietic celloutput of RUNX1.

The marker gene CD31, which is expressed primarily on endothelial cellsduring development, was very highly expressed in the primitive waveearly time point. However, following the primitive wave, its expressionfollowed that of DEAB, suggesting that these primitive wave endothelialcells are maintained in the culture but are reduced with the RAinhibitor DEAB (FIG. 6L).

SCL is a marker of both primitive and definitive hematopoiesis.Increased SCL expression with DMSO compared to DEAB in the early timepoints (FIG. 6M, Day 8-12) agrees with the above assessment of DMSOhaving more primitive wave hematopoiesis than DEAB at these time points.However, increased SCL expression was observed with DEAB compared toDMSO at later time points (FIG. 6M, Day 15), indicating definitivehematopoiesis with DEAB. See Zhang Y, Payne K J, Zhu Y, Price M A,Parrish Y K, Zielinska E, Barsky L W, Crooks G M, “SCL expression atcritical points in human hematopoietic lineage commitment,” Stem Cells.2005 June-July; 23(6):852-60.

Based on these marker expression profiles in combination with the invitro differentiation data (functional and FACS where we demonstratesignificant numbers of cells with definitive hematopoietic phenotype(CD45+, CD43+), and generate lymphoid lineages, see at least Examples1-3 above and Example 5 below), the data strongly suggest thatdefinitive hematopoietic cells are the predominant hematopoietic cellsgenerated in our system with the RA inhibitor DEAB.

To confirm the RA signaling inhibition using DEAB, we measured thelevels of expression of known gene targets of RA. We detected reducedlevels of RARa (FIG. 6N, Day 8) and RARb (FIG. 6O, Day 8) expression,demonstrating inhibition of the feed forward regulation of RA signaling.Similarly, reduction of the known RA target, CD38, was also confirmed byPCR (FIG. 6P), confirming the reduced CD38 expression seen by FACS asdescribed earlier.

Example 5 Differentiation into Lymphocytes

Hematopoietic progenitors and phenotypic stem cells obtained bydifferentiating iPS cells using the basal medium supplemented with DEABat 10 uM were differentiated into lymphoid cells (B, NK, T) as describedin Nozad Charoudeh H, Tang Y, Cheng M, Cilio C M, Jacobsen S E, SitnickaE, “Identification of an NK/T cell-restricted progenitor in adult bonemarrow contributing to bone marrow- and thymic-dependent NK cells.,”Blood. 2010 July 15:116(2):183-92. Briefly, either CD45+, CD43+, CD34+,7AAD− hematopoietic progenitors, or CD45+, CD43+, CD34+, CD38−, CD90+,CD45RA− phenotypic HSCs were plated side by side with cord bloodisolated hematopoietic progenitors at 20-40 cells per well ontopreviously established, approximately 80% confluent stroma cellmonolayers of OP9 and OP9delta feeder lines for B-cell and T-celldifferentiation respectively in 48-well plates in 1 mL of Opti MEM plusGlutaMax (Life Technologies, Carlsbad Calif.) containing 10% fetal calfserum (FCS; Sigma-Aldrich), 1% penicillin/streptomycin (Sigma-Aldrich),1% 2M 2-mercaptoethanol (Sigma-Aldrich) supplemented with cytokines withfinal concentrations: FLT3 ligand (FL, 25 ng/mL), interleukin-7 (IL-7,20 ng/mL), KIT ligand (KL, 25 ng/mL), IL-15 (25 ng/mL), and IL-2 (50ng/mL). Cells were cultured at 37° C. for 14 to 21 days, and half of thecoculture medium was replaced weekly. Between 14 and 21 days afterdifferentiation, cells were harvested and analyzed by FACS side by sidewith the cord blood isolated progenitors differentiated to B, NK, and Tcells under the identical method as described above for pan-lymphoidlineage (CD33−, FIGS. 7A and 7B) and lymphoid committed progeny (B cell(CD19+, CD10+; FIGS. 7C and 7D), T cell (CD3+, CD4+; FIGS. 7I and 7J)(CD5+, CD7; FIGS. 7E and 7F), and NK cell (CD56+, CD16+; FIGS. 7G and7H)). The results show that the HSCs generated as described above arecapable of differentiating to each type of tested lymphocyte.

Example 6 LY222880

We conducted experiments to determine whether the p38 inhibitor LY222880could further increase hematopoietic progenitor and phenotypic stem celloutputs by acting in concert with DEAB when added to DifferentiationMedium 1. Human ES and iPS cells were differentiated to progenitors andphenotypic stem cells as described, except 100 nM of LY222880 was addedto Differentiation Medium 1. Cells were then harvested as described andsubjected to flow cytometry and FACS analysis using fluorescentlylabeled antibodies for CD45, CD43, CD34, CD38, CD90, CD45RA asdescribed. Results show that the phenotypic hematopoietic stem cellphenotype (CD45+, CD43+, CD34+, CD38−. CD90+, CD45RA−) was increased bynearly 20% over the DEAB-containing Differentiation Medium 1 control,whereas levels of the intermittent progenitor phenotype (CD45+, CD43+,CD34+, CD38−) were unaffected (FIG. 8A). Conversely for iPS cells, theaddition of 100 nM of LY222880 increased numbers of all cells with theprogenitor and phenotypic stem cell phenotypes (FIG. 8B). These resultssuggest that progenitor and/or phenotypic stem cell yields can befurther increased when using LY222880 in concert with DEAB in ourdifferentiation medium and protocol.

Example 7 Norepinephrine

We conducted experiments to reveal if human embryo AGM region-harvestedcells generate neurospheres in culture to confirm the presence of neuralcrest cells in the AGM region. We further completed some experiments toidentify a component secreted from the neural crest cells that may beinvolved in definitive hematopoiesis. We harvested human embryo AGMregion tissue and, using fluorescent labeled antibodies specific forneural crest cells, were able to identify the presence of neural crestcells in the AGM hematopoietic niche. This suggested that neural crestcells play a role in hematopoiesis. Hypothesizing norepinephrine as apotential effector secreted from the neural crest cells, we tested itfor its ability to further increase progenitor and phenotypic stem cellyields when added to Differentiation Medium 1 at a concentration of 10uM or 300 uM. Human ES and iPS cells were differentiated to progenitorsand phenotypic stem cells with Differentiation Medium 1 as described.The only difference is that 10 uM or 300 uM of norepinephrine was addedto Differentiation Medium 1. Cells were then harvested as described andsubjected to flow cytometry and FACS analysis using fluorescentlylabeled antibodies for CD45, CD43, CD34, CD38, CD90, CD45RA asdescribed. The results of this experiment reveal statisticallysignificant increases in fold improvement of numbers of phenotypichematopoietic stem cells (CD45+, CD34+, CD43+, CD38−, CD90+, CD45RA−,FIG. 9C) whereas insignificant improvements were observed in the totalviable blood cells (CD45+, FIG. 9A) and the intermediate cells (CD45+,CD43+, CD34+, CD38−, FIG. 9B). The results demonstrate thatnorepinephrine has an effect on the most primitive fraction ofhES-derived hematopoietic cells (150% increase in HSC number) and oncolony forming ability (data not shown). These data suggest thatnorepinephrine is an effector secreted from neural crest cells. Giventhe verified presence of neural crest cells in the AGM niche, we areconfident that these cells play a role in establishing definitivehematopoiesis in the developing embryo. As such, long term engraftmentand reconstitution ability of hematopoietic progenitors and phenotypicstem cells produced from iPS and ES cells differentiated withDifferentiation Medium 1 plus norepinephrine as described above arecurrently underway. The engraftment and reconstitution studies will beconducted as described by the protocol outlined above in the Materialsand Methods.

Example 8 Additional Molecules

We have completed a number of studies to establish other molecules thatmay further improve the yields and phenotypes of hematopoietic cellsderived using Differentiation Medium 1. Tested compounds were Cripto,LY2228820, valproic acid (VPA), sodium selenite, and butein. Data forthese are defined below. In all instances, each molecule below wassupplemented into Differentiation Medium 1 to study their effect inconcert with DEAB. All control experiments for these molecules werecells differentiated using Differentiation Medium 1 (which alreadycontains DEAB). The data we report below for these molecules illustrateshow they may improve the yield of hematopoietic progenitors andphenotypic stem cells measured by FACS, flow cytometry, and CFU assays.It is important to note when interpreting these results that thedefinitive hematopoietic stem cells in vivo do exist in a quiescent,non-proliferative state during part of their life cycle. As such,molecules that do not dramatically improve CFU counts of these cells maystill contribute to improving HSC quiescence, thought to be necessaryfor high levels of engraftment. The in vivo engraftment studies toconfirm this for these molecules are underway. See FIG. 14 for the CFUdata. This data taken in context with FIGS. 10A, 10B, 11A, 11B, 12A,12B, 13A, and 13B showing increased fraction of phenotypic HSCs suggestthat cells produced with Differentiation Medium 1 supplemented withthese compounds reduces stress and increases production of quiescentHSCs.

As shown in FIGS. 10A and 10B, LG101506 (1 uM) when used in theDifferentiation Medium 1 medium increased the total number of cells(similar to the DEAB results) for both pluripotent lines (ES, iPS),indicating that this also increases the total output of CD90+ cells.Interestingly, the combination of DEAB with LG101506 further increasedthe numbers of CD38− cells compared to either compound alone, andgenerates the highest numbers of phenotypic HSCs.

It has been shown that DS DNA breaks reduce transplant efficiency ofrepopulating HSCs. In vitro derived blood from pluripotent stem cell hashigh levels of DNA damage.

Inhibition of DNA damage and the DNA damage response, from reactiveoxygen species (ROS) or other sources of damage (radiation, chemicaletc) in pluripotent stem cell differentiation systems will decrease DNAdamage and increase hematopoietic cell number, repopulating ability andincrease the differentiation potential of the cells (i.e. expansion ofdownstream lineages). See Yahata et al. Blood, 2011, 118(11):2941-50).We show that simple culture differentiation of pluripotent cells resultsin many double strand (DS) DNA breaks as marked by gH2AX staining.Adding sodium selenite (100 nM) and butein (10 uM) independently toDifferentiation Medium 1 increased enzyme activity for protectionagainst reactive oxygen species (ROS). Each of sodium selenite (100 nM)and butein (10 uM) independently added to Differentiation Medium 1 alsoincreased CD34+, CD38− cells as well as total cells. See FIGS. 11A and11B for sodium selenite and FIGS. 12A and 12B for butein. These studiesare being expanded into dose response experiments from which we expectto identify optimal concentrations.

From the pluripotent stem cells differentiated with DifferentiationMedium 1 supplemented with 1 mM of valproic acid, we see that all CD34+CD38− cells were 100% CD90+. The total number of cells was alsoincreased. See FIGS. 13A and 13B. These results are being expandedfurther into a dose response study from which we expect to identify theoptimal concentration. Engraftment studies as described are also beingconducted.

Example 9 Pluripotent Stem Cell Derived Hematopoietic CellTransplantation

We have confirmed short-term engraftment and reconstitution ability ofhPSC-derived HSCs produced as described in Example 1, by injecting theminto sublethally irradiated (325 rad) NOD.Cg-Prkdc^(scid)Il2rg^(tmlWjl)/SzJ mice (JAX, Bar Harbor, Me., www.jax.org) via tailvein injections, intrafemoral injection, or cardiac injection. Wetransplanted NOD.Cg-Prkdc^(scid)Il2rg^(tmlWjl)/SzJ mice with 3×10⁶unsorted cells or 5000 FACS sorted phenotypic HSCs (CD45+, CD43+, CD34+,CD38−, CD90+, CD45RA−) from differentiations using DifferentiationMedium 1 medium not including with DEAB (Control) and DifferentiationMedium 1 medium including DEAB (DEAB HSCs). We analyzed the red bloodcell-lysed, mononuclear fraction of peripheral blood samples collected 4weeks post-transplant for the percentage of cells exhibiting thepan-hematopoietic human blood cell marker CD45+ by FACS or flowcytometry. Typical published values range from 0.1%-1.0%, includingthose obtained from our control where DEAB was not used to generate theHSCs (0.34%±0.1%). (See Wang et al. The Journal of ExperimentalMedicine, 2005, 201(10):1603-14; Lu et al. Experimental hematology,2009, 37(8): 924-36; Tian et al. Stem cells, 2009, 27(11): 2675-85;Ledran et al. Cell stem cell, 2008, 3(1):85-98; Bhatia, M.,Hematopoietic development from human embryonic stem cells.Hematology/the Education Program of the American Society of Hematology.American Society of Hematology. Education Program, 2007: p. 11-6; andBhatia, M. Annals of the New York Academy of Sciences, 2007,1106:219-22.) We demonstrated 2-fold higher engraftment (2.2%±0.2%,n=4), suggesting that HSCs differentiated from pluripotent stem cellsusing Differentiation Medium 1 medium containing DEAB significantlyimproves engraftment outcome upon transplantation and is ideal fortesting and incorporating additional factors to improve hPSC-derived HSCengraftment and reconstitution to levels required for therapeutics.

Further engraftment studies are underway with the HSC cells generated asdescribed above. We predict that phenotypic HSC cells produced withDifferentiation Medium 1 (containing DEAB) supplemented with any of oneor more of Cripto, LY222820, valproic acid (VPA), sodium selenite,butein, norepinephrine, and LG101506 will generate cells suitable forsuccessful in vivo engraftment.

We claim:
 1. A differentiation medium comprising a retinoic acidsignaling inhibitor, an antioxidant, and BMP4.
 2. The differentiationmedium of claim 1 further comprising one, all, or any subcombination ofcomponents selected from the group consisting of a stimulant ofprostaglandin E₂ pathway, TPO, VEGF, SCF, FLT-3, EPO, and TGFβ1.
 3. Thedifferentiation medium of claim 1 further comprising one, all, or anycombination of components selected from the group consisting of a p38MAPK inhibitor, a beta adrenergic receptor agonist, a cell cycleinhibitor, an RXR agonist, Cripto, and a chromatin remodeler.
 4. Thedifferentiation medium of claim 3 wherein the p38 MAPK inhibitor isLY2228820, the cell cycle inhibitor is rapamycin, the RXR agonist isLG101506, and the chromatin remodeler is valproic acid.
 5. Thedifferentiation medium of claim 3 wherein the beta adrenergic receptoragonist comprises one, all, or any combination of components selectedfrom the group consisting of norepinephrine, epinephrine, salmeterol,and isoproterenol.
 6. The differentiation medium of claim 1 wherein theantioxidant comprises one, all, or any combination of componentsselected from the group consisting of sodium selenite, butein,glutathione, and N-acetyl cysteine.
 7. The differentiation medium ofclaim 1 wherein the retinoic acid signaling inhibitor comprises DEAB. 8.A method of differentiation comprising exposing a cell to adifferentiation medium as described in claim
 1. 9. The method of claim 8wherein the cell comprises a pluripotent stem cell and wherein theexposing comprises exposing for a time and under conditions sufficientto generate a hematopoietic stem cell.
 10. The method of claim 9 whereinthe hematopoietic stem cell is capable of further differentiating into amyeloid cell and is also capable of further differentiating into alymphoid cell.
 11. The method of claim 9 further comprisingdifferentiating the hematopoietic stem cell into a cell selected fromthe group consisting of a myeloid cell and a lymphoid cell.
 12. Themethod of claim 11 wherein the differentiating the hematopoietic stemcell is performed in vitro.
 13. The method of claim 11 wherein thedifferentiating the hematopoietic stem cell is performed in vivo. 14.The method of claim 8 wherein the cell is a human cell.
 15. The methodof claim 8 wherein the exposing comprises initially exposing the cell tothe differentiation medium by adding a volume of the differentiationmedium to an existing volume of non-differentiation medium to generate amixed medium, exposing the cell to the mixed medium, and subsequentlyreplacing substantially all the mixed medium after about 12-48 hoursfrom the initial exposing with a fresh volume of the differentiationmedium.
 16. The method of claim 15 wherein after the replacingsubstantially all the mixed medium the exposing further comprises addinga fresh volume of the differentiation medium only to an existing volumeof the differentiation medium, wherein the existing volume comprisesmedium exposed to the cell for a period of at least two hours.
 17. Themethod of claim 8 wherein the cell comprises an intact embryoid bodyformed for a period of at least 7 days prior to the exposing.
 18. Themethod of claim 17 wherein the exposing comprises exposing the intactembryoid body to the differentiation medium for a period of at least 5days prior to plating the embryoid body, and then plating the embryoidbody.
 19. The method of claim 18 wherein the exposing, after theplating, further comprises adding a fresh volume of the differentiationmedium only to an existing volume of the differentiation medium withoutremoving any of the existing volume of the differentiation medium,wherein the existing volume comprises medium exposed to the cell for aperiod of at least 15 minutes.
 20. The method of claim 8 wherein theexposing comprises exposing the cell to the differentiation medium inthe absence of feeder cells.
 21. A method of engrafting a blood cell ina human subject comprising administering a hematopoietic stem cell or acell differentiated therefrom to the subject, wherein the hematopoieticstem cell is generated by exposing a cell to a differentiation medium asdescribed in claim 1.